Ctivity-dependent gene transcription. We initially demonstrated by Western blotting that MeCP
Ctivity-dependent gene transcription. We very first demonstrated by Western blotting that MeCP2 T308A KI mice and their wild-type littermates express equivalent levels of MeCP2 protein. This indicates that the T308A mutation will not alter the stability of MeCP2. Moreover, we confirmed by Western blotting with anti-MeCP2 phospho-T308 antibodies that the MeCP2 T308A KI neurons lack T308 CCR4 web phosphorylation (Supplementary Fig. 10a ). We also demonstrated by chromatin immunoprecipitation with anti-MeCP2 antibodies that the T308A mutation will not affect MeCP2 binding to DNA (Supplementary Fig. 10d), and by peptide pull-down experiments (Fig. 2b) and co-immunoprecipitation of MeCP2 and NCoR from forebrain extracts (Supplementary Fig. 10e), that the T308A mutation will not disrupt the all round binding of MeCP2 for the NCoR complex. These findings suggest that any abnormality that we detect in gene transcription in MeCP2 T308A KI mice may possibly be attributed for the loss in the phosphorylation-dependence from the interaction of MeCP2 with the NCoR complex as an alternative to to a decrease in MeCP2’s expression, binding to DNA, or general ability to interact with NCoR. We assessed the effect of your MeCP2 T308A mutation on activity-dependent gene transcription straight by exposing cultured neurons derived from wild-type and MeCP2 T308A KI mice to elevated levels of KCl and monitoring activity-dependent gene expression by RT-PCR (Fig. 3a). We discovered that membrane depolarization induces Arc, Fos, Nptx2, and Adcyap1 mRNA expression equivalently in wild-type and MeCP2 T308A KI neurons indicating that the signaling apparatus that conveys the membrane depolarization/ calcium signal towards the nucleus to activate gene transcription functions ordinarily in MeCP2 T308A KI neurons. By contrast, membrane depolarization induces considerably much less Npas4 in MeCP2 T308A KI neurons than in wild-type neurons. Earlier studies have shown that Npas4 expression is induced upon membrane depolarization of excitatory neurons and thatNature. Author manuscript; available in PMC 2014 July 18.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptEbert et al.PageNPAS4 promotes the LTB4 Compound development of inhibitory synapses on excitatory neurons18, a procedure that has been discovered to become abnormal in RTT19. NPAS4 is usually a transcription factor which has been recommended to regulate inhibitory synapse quantity by activating expression of Bdnf18. Therefore, we asked if Bdnf may also be impaired in T308A KI neurons in comparison to wildtype neurons. There is a trend towards decreased induction of Bdnf mRNA in T308A KI neurons in comparison with wild-type neurons. We also observed an attenuation of light induction of Npas4 and Bdnf inside the visual cortex of dark-reared T308A KI in comparison with wild-type mice but no statistically substantial difference in Arc, Fos, Nptx2, and Adcyap1 mRNA expression in these two strains of mice (Fig. 3b). This suggests that the decrease in activity-dependent Npas4 and Bdnf expression in T308A KI in comparison with wild-type mice occurs in vivo and could in principle contribute to neural circuit defects that occur in RTT. These findings are consistent having a model in which activity-dependent phosphorylation of MeCP2 T308 leads to decrease inside the association with the NCoR co-repressor complex using the repressor domain of MeCP2, hence facilitating activity-dependent Npas4 transcription and also the subsequent activation of Bdnf transcription. Even so, given that MeCP2 binds broadly across the genome, we can not.
