Intense investigation has focused on determining how mutations of MECP2
lead to RTT and related neurological disorders. The postnatal time course of RTT symptom onset together with the synaptic defects observed in Mecp2
mutant mice have led to the hypothesis that RTT is a disorder of experience-dependent synapse maturation. However, the devastating consequences of loss or over-expression of MeCP2 on cell and organismal health have made it difficult to assess whether defects in experience-dependent synaptic and cognitive development arise directly from, or are indirect consequences of, loss of MeCP2 function. Indeed, careful observation of individuals with RTT has suggested that different mutations in MECP2
can lead to distinct cognitive and clinical sequelae (Neul et al., 2008
), suggesting that MeCP2 has a number of discrete roles in the development of the nervous system.
The discovery that experience induces the phosphorylation of MeCP2 at S421 in the brain revealed a mechanism by which neuronal activity might modulate MeCP2 function, and has provided a molecular handle to dissect the activity-dependent and -independent functions of MeCP2. In the present study we eliminated the neuronal activity-dependent phosphorylation of MeCP2 at S421 in vivo without otherwise affecting MeCP2 expression. By studying these MeCP2 S421A mice we find that MeCP2 S421 phosphorylation is required for the normal development of neuronal dendrites and inhibitory synapses in the cortex, demonstrating the importance of the activity-dependent regulation of MeCP2 for the establishment of appropriate connectivity in the nervous system. In addition, we find that loss of MeCP2 S421 phosphorylation results in defects in behavioral responses to novel versus familiar mice or objects, indicating that activity-dependent MeCP2 phosphorylation regulates aspects of cognitive function. Based on these findings, we propose that the disruption of MeCP2 phosphorylation at S421 contributes to the cognitive impairments observed in RTT and other MECP2-dependent disorders.
Compared to the effects of deleting Mecp2, mutating MeCP2 S421 to an alanine has a relatively mild effect on nervous system development. For example, the gross motor abnormalities, body weight dysregulation, seizures, and certain learning and memory defects observed in the MeCP2 knockout appear not to rely on the activity-dependent phosphorylation of MeCP2 at S421. This could suggest that aspects of MeCP2-regulated neuronal function rely on neuronal activity-independent development processes. Alternatively, it is possible that other stimulus-dependent MeCP2 modifications (D. Ebert and M. Greenberg, unpublished results) may function either singly or in combination to regulate MeCP2-dependent neuronal responses.
It has been proposed, based on mass spectrometry analysis (Tao et al., 2009
), that phosphorylation of MeCP2 also occurs at serine 424 (S424). A recent study reports that the mutation of both MeCP2 S421 and S424 to alanines in mice results in alterations in hippocampal learning and synapse biology as well as changes in MeCP2 binding and dysregulation of a small number of candidate genes examined (Li et al., 2011
). The phenotypes reported in these mice are similar to the phenotypes observed when MeCP2 is over-expressed in mice (Chao et al., 2007
; Collins, 2004
) raising the possibility that the mutation of S424 to alanine leads to enhanced MeCP2 expression or activity.
In an effort to determine if neuronal activity induces the phosphorylation of MeCP2 S424 we have generated anti-phospho-S424 MeCP2-specific antibodies, but we have been unable to detect increased phosphorylation of MeCP2 S424 in response to neural activity in vitro (KCl depolarized vs unstimulated cortical cultures,) or in vivo (kainate seized vs unseized brain) (D. Ebert and M. Greenberg, unpublished results). While it remains possible that MeCP2 S424 is phosphorylated constitutively or in response to other stimuli, we have restricted our analysis to the verified activity-dependent phosphorylation of MeCP2 at S421, allowing us to unambiguously relate the phenotypes we observe in MeCP2 S421A mice to activity-dependent MeCP2 phosphorylation.
Our observations using MeCP2 S421A mice reinforce the importance of in vivo
models for studying the role of neuronal activity in nervous system development and function. Previous in vitro
studies suggested a model in which, in the absence of neuronal activity, MeCP2 is bound to the promoters of activity-regulated genes such as Bdnf
to repress their transcription (Chen et al., 2003
; Martinowich, 2003
; Zhou et al., 2006
). Membrane depolarization-induced S421 phosphorylation was proposed to lead to reduced binding of MeCP2 at these activity-dependent promoters, relieving repression and allowing for gene activation. If this model were correct we would predict that neurons from MeCP2 S421A mice might demonstrate a defect in the induction of Bdnf
or other activity-regulated genes. Instead, we found that the activity-dependent expression of Bdnf
and other neuronal activity-dependent genes was not detectably dysregulated by loss of MeCP2 phosphorylation at S421, suggesting that this model of activity-dependent MeCP2 function must be revised.
To reassess the molecular function of MeCP2 and its regulation by neuronal activity, we turned to ChIP-Seq to examine the binding profile of MeCP2 genome-wide. Recent work from brain had suggested that MeCP2 binds broadly across the genome (Skene et al., 2010
). By demonstrating that MeCP2 is highly enriched throughout the genome in both the brain and dissociated cortical cultures that contain very few glial cells, we exclude the possibility that the broad binding of MeCP2 observed in the brain arises as a result of heterogeneous contributions from neuronal and glial populations. Consistent with this recent study (Skene et al., 2010
), the pattern of binding we detect in brain and neurons suggests that MeCP2 binds preferentially to methylated DNA (i.e. reduced binding at TSS sites, increased binding at repeat DNA). However, MeCP2 binding is not limited to methylated loci, as we note a high level of signal in MeCP2 ChIP assays from brain and cultured neurons at sites where DNA methylation is presumably very low (e.g. the TSS for the highly-expressed Myc
gene), or devoid of CpG residues over long stretches. Interestingly, the ChIP profile of MeCP2 in E16 + 7 DIV cortical cultures is more flat than that found in the brains of 7-week-old mice (e.g. Figure S4A
), suggesting that changes in DNA methylation or MeCP2 expression levels during nervous system development may lead to an increase in the dynamic range of the MeCP2 binding profile.
Taken together, our ChIP data allow us to conclude that MeCP2 is bound throughout the neuronal genome in a pattern similar to that of a histone protein. Several studies have demonstrated that MeCP2 binds to the linker DNA between nucleosomes in vitro
similarly to linker histone H1 (Ghosh et al., 2010
; Nan et al., 1997
), and that in vivo
histone H1 levels are up-regulated in the MeCP2 knockout brain (Skene et al., 2010
). Our data is consistent with a model in which MeCP2 takes the place of H1 molecules throughout the neuronal genome, functioning on a global scale to modulate chromatin structure.
By examining genome-wide profiles of MeCP2 before and after neuronal stimulation, we have assessed the potential for dynamic regulation of MeCP2 binding by activity-dependent phosphorylation. Under the conditions used for these experiments, S421 phosphorylation is induced on a substantial fraction of MeCP2 molecules, yet we do not detect changes in the profile of MeCP2 binding across the genome. Because of the broad distribution of MeCP2, low read coverage limits our power to detect discrete regions where binding may be lost. However, using more sensitive ChIP-qPCR at multiple candidate activity-dependent loci we are unable to detect stimulus-dependent changes in binding. Consistent with this finding, seizure-induced increases in neuronal activity do not detectably alter MeCP2 binding at the activity-regulated promoter of Bdnf (H. Gabel, B. Kinde and M. Greenberg, unpublished observations). While it remains possible that a small number of discrete sites experience changes in binding or that there is a subtle change in global binding within the variability of our experiments, our data suggest that a stimulus capable of robustly inducing MeCP2 S421 phosphorylation is not sufficient to cause MeCP2 dissociation from the genome. Furthermore, because our stimuli induce the expression of Bdnf and other activity-regulated genes, dissociation of MeCP2 from the DNA is not strictly required for transcriptional induction of these genes. Instead it appears that neuronal activity induces the phosphorylation of MeCP2 molecules that remain bound to the genome, serving to modulate MeCP2 function in situ.
Given the histone-like binding of MeCP2 to the neuronal genome, we considered that the phosphorylation of MeCP2 S421 could function in a manner analogous to a histone modification. While studies of pan-histone genomic binding profiles have provided important information about chromatin structure, ChIP analysis of specific histone modifications has led to a rich understanding of the localization and dynamics of these modifications, providing insight into their function in the modulation of gene expression (Zhou et al., 2011
). As a first step toward understanding where post-translational modifications of MeCP2 occur on the genome, we performed ChIP analysis using a specific pS421 MeCP2 antiserum. We demonstrate that the neuronal activity-induced phosphorylation of S421 is evenly distributed across MeCP2 molecules bound to the genome. We estimate the percentage of MeCP2 phosphorylated at S421 in response to neuronal stimulation (2 hr KCl depolarization) to be 10–30%. If one MeCP2 molecule is bound every two nucleosomes as demonstrated by (Skene et al., 2010
), and phosphorylation is evenly distributed across MeCP2 molecules, then an independent phosphorylation event is occurring approximately every 900–3000 bp. Thus, pS421 MeCP2 is likely to be extremely common across the genome, and has the potential to affect chromatin at a genome-wide scale. These findings suggest that instead of regulating specific target genes, MeCP2 S421 phosphorylation likely plays a more global role in modulating the response of neuronal chromatin to activity.
While many histone modifications have been found in discrete loci, genome-wide phosphorylation of histone H3 (e.g. H3S10) and histone H1 are thought to facilitate mitotic chromosomal rearrangements in non-neuronal cells (Happel and Doenecke, 2009
; Nowak and Corces, 2004
). This precedent suggests that the global phosphorylation of MeCP2 may alter chromatin compaction states throughout the nucleus or facilitate nuclear reorganization events that have been reported to occur in response to neuronal activity (Wittmann et al., 2009
). As a result, the phosphorylation of MeCP2 S421 may play a role in fine-tuning aspects of gene expression that are not readily detected with standard measures of gene expression. Alternatively, the global phosphorylation of MeCP2 S421 could collaborate with locus-specific modifications of MeCP2: S421 phosphorylation would facilitate transcriptional processes specifically gated by other stimulus-dependent modifications of MeCP2. Either of these models could explain why we observe phenotypes consistent with defects in experience-dependent neuronal development upon loss of MeCP2 S421 phosphorylation, but are unable to detect significant changes in the expression of individual genes.
Together with recent analysis (Skene et al., 2010
), our data support a conceptual shift in the understanding of the function of MeCP2 in neurons. Instead of acting solely as a repressor to regulate gene expression through targeted, dynamic binding to chromatin, it may be more appropriate to consider MeCP2 as a constitutive component of neuronal chromatin. The idea that MeCP2 has many functions is consistent with the discovery of multiple, independently occurring phosphorylation events on MeCP2 (Huttlin et al., 2010
; Tao et al., 2009
; Zhou et al., 2006
) (D. Ebert and M. Greenberg, unpublished observations) and the finding that total loss or over-expression of MeCP2 leads to subtle changes in the expression of thousands of genes rather than derepression of a discrete subset of target genes. Just as different histone modifications can correlate with independent and often opposing effects on gene expression, the different modifications of MeCP2 may have distinct influences on chromatin at sites where they occur. The experiments presented here demonstrate that it is possible to perform modification-specific ChIP analysis of MeCP2 to gain insight into its global role. Future studies employing this approach may indicate where additional phosphorylation events occur on the genome and provide insight into how they modulate MeCP2 function.
By revealing the functional role of MeCP2 S421 phosphorylation, the MeCP2 S421A mouse demonstrates the utility of an in vivo approach for testing hypotheses regarding activity-dependent regulation of MeCP2. Moreover, the phenotypes observed in the MeCP2 S421A mice provide in vivo evidence that the stimulus-dependent modification of a chromatin regulator is required for nervous system development and function. Additional knock-in mutations to disrupt other activity-dependent modifications of MeCP2 may provide useful models for further study of stimulus-dependent regulation of MeCP2 in vivo, and should yield insight into the role of the environment in regulating neuronal chromatin function. Finally, given the critical importance of MeCP2 for nervous system development, future experiments to understand activity-dependent MeCP2 regulation will not only serve to deepen our understanding of stimulus-dependent chromatin biology, but should also provide therapeutic insight into RTT and other neuropsychiatric disorders.