Although we detected more than 300 genes whose check details expression levels changed at least 3-fold in response to extracellular KCl, we found no significant differences in the number, extent, or time course of induction of activity-dependent mRNAs between the wild-type and MeCP2 S421A cells (Figure S5). We also used Mouse Gene 1.0 ST microarrays to assess mRNA levels in the visual cortex of postnatal day 16–17

wild-type and MeCP2 S421A knockin brains, a time point and brain region where MeCP2 is phosphorylated at S421 (Figure S6). An analysis of transcript levels using either individual probes within specific gene regions or groups of probes across individual genes revealed no detectable mRNA dysregulation in the developing MeCP2 S421A mouse brain (data not shown). It remains possible that S421 phosphorylation has more subtle effects on the magnitude or timing of activity-dependent gene transcription. Alternatively, MeCP2 S421 phosphorylation may control aspects of transcription (e.g., initiation, elongation, termination, or the rate of transcription) or chromatin remodeling

(e.g., histone modification and compaction or DNA methylation) that are not detected by measuring steady-state mRNA levels. It is also possible that other mechanisms of gene regulation largely compensate in the absence of MeCP2 S421 phosphorylation. AP24534 nmr To further investigate how S421 phosphorylation might affect MeCP2 function, we employed ChIP-Seq to examine where across the genome MeCP2 becomes newly phosphorylated at S421 in response to neuronal activity. We hypothesized that if pS421 MeCP2 controls a specific step in the process of activity-dependent gene transcription, then this phosphorylation event would be expected to occur at select regions of the genome (e.g., the promoters, enhancers, or exons of activity-dependent genes). We first sought to establish the utility of our anti-pS421 MeCP2 specific antiserum for ChIP analysis (Figure 1A and Figure S4B). We immunoprecipitated pS421 MeCP2 from unstimulated or KCl-depolarized neurons and from the brains

of wild-type or MeCP2 S421A knockin mice. qPCR analysis of several loci demonstrated that the anti-pS421 MeCP2 antibody specifically recognizes the phosphorylated form of MeCP2 in ChIP assays: pS421 MeCP2 was found to be bound at all sites tested in membrane-depolarized neurons but not in unstimulated Calpain neurons where the level of MeCP2 S421 phosphorylation is quite low (Figure 7A), and was enriched in wild-type brain compared to MeCP2 S421A brain (Figure 7B). Moreover, the pS421 MeCP2 ChIP signal was competed away if the anti-pS421 MeCP2 antiserum was preincubated with the phospho-peptide antigen used to generate the antibody (Figure S4C). To determine where along the genome MeCP2 S421 becomes phosphorylated in response to neuronal activation, we performed high-throughput sequencing of pS421 MeCP2 ChIP DNA isolated from cultured cortical neurons treated with 55mM KCl for 2 hr.