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. 2023 Oct 23;120(44):e2310344120. doi: 10.1073/pnas.2310344120

Fig. 1.

Fig. 1.

Activity-dependent modifications of MeCP2 and generation of MeCP2 quadruple knock-in (QKI) mice. (A) Top: Quantitative assessment of stimulus-responsive MeCP2 post-translational modifications. Neuronal activity was induced in vivo with injection of KA for 1 h or saline (control) in mice, after which hippocampi were dissected. Cultured mouse cortical neurons were either left untreated (control) or stimulated at in vitro day 7 with BDNF (50 ng/mL) or KCl (55 mM) for 30 min to induce depolarization. After induction, MeCP2 protein was isolated using immunoprecipitation, after which samples underwent quantitative mass spectrometry to assess post-translational modifications. Phosphorylation at residues S86, S274, T308/T311, and S421 of MeCP2 were found to be induced by neuronal activity. Bottom: Schematic depiction of the MeCP2 protein showing the characterized methyl-DNA-binding domain (MBD) and transcriptional repression domain (TRD) as well as the location of the four sites of activity-responsive phosphorylation targeted for mutation. (B) Sanger sequencing confirms the expected base substitutions in QKI mice. (C) Western blotting of dLGN and visual cortex from P27-P32 mice with the indicated antisera confirms the loss of MeCP2 S421 phosphorylation in QKI mice without affecting the total levels of MeCP2 protein (n = 3 mice). Equal amounts of protein lysates from WT and QKI brain tissue were loaded on a denaturing protein gel, followed by western blotting with an MeCP2-S421 phospho-specific antiserum, an antibody against total MeCP2, and an antibody specific for β-actin as a loading control. (D) Quantification of western blots shown in (C). Levels of MeCP2 phosphorylated-S421 are significantly reduced in QKI dLGN and visual cortex compared to WT animals (Left). In contrast, levels of total MeCP2 protein are unaffected in QKI mice in both dLGN and visual cortex (Middle) (n = 3 mice). Protein levels in each lane were normalized to the corresponding β-actin control (Right). *P < 0.05, unpaired, two-tailed Student’s t test.