Figures & data
Figure 1. MeCP2 is acetylated by p300 at Lys-464. (A) The mouse MeCP2 isoform 1 (MeCP2e1-FLAG) expression vector was transfected in HEK-293 cells, and immunoprecipitated samples were probed with anti-pan-acetyl-lysine antibody. (B) HA-tagged p300, but not HA-tagged CBP, increases acetylation of MeCP2 at lysine 464 in HEK-293 cells. FLAG-MeCP2e1 WT or FLAG-MeCP2e1 K464 point mutant were co-expressed together with HA-tagged p300 or HA-tagged CBP in HEK-293 cells. (C) Level of acetylated Lys-464/ MeCP2 was set as 1; p300 is capable to significantly increase the amount of acetylated MeCP2. Blots are representative of three independent experiments (n=3). Student's t test. Values represent means ± SD. *p<0.02 **p<0.01 (D) Anti-acetyl MeCP2-K464 (AcMeCP2) antibody recognizes recombinant GST-MeCP2e1 acetylated by p300, not GST-MeCP2e1 alone or GST-empty (GST-EV). (E) In vitro acetylation assay. Signal for acetylated MeCP2 is detected in the presence of p300. (F-G) MeCP2 interacts with wild type SIRT1, but not with the catalytically inactive mutant H363Y (HEK-293). (H) SIRT1 wild type (WT) or the catalytically inactive mutant (H363Y) were co-expressed in HEK-293 cells together with MeCP2e1. After MeCP2 immunoprecipitation the level of acetylated Lys-464 was monitored in the presence of SIRT1 WT or H363Y mutant. (I) Level of acetylated Lys-464/ MeCP2 was set as 1. Co-expression of SIRT1 WT, but not the catalytically inactive one (H363Y), decreases K464 acetylation. Blots are representative of three independent experiments (n=3). Student's t test *p<0.02 **p<0.01. t test n=3. Values represent means ± SD. *p<0.02 **p<0.01
Figure 2. SIRT1 controls MeCP2 acetylation in vitro and in primary neurons. (A) SIRT1 deacetylates MeCP2 in vitro. Immunoprecipitated MeCP2 was incubated with purified recombinant SIRT1 in deacetylation buffer that contained NAD+, TSA, EX527, or nicotinamide. MeCP2 acetylation at lysine 464 (AcMeCP2) was monitored by Western analysis. The experiments were repeated three times (B) Immunofluorescence of cortical neurons (DIV5) after EX527 treatment (6 hours). Bar=65 mm (C) Intensity of fluorescence shown in panel B is evaluated as pixels/µm2 (Materials and Methods) Student's t test (n=3) Values represent means ± SD. *p<0.05, **p<0.01, ***p<0.001. (D) Immunofluorescence of cortical neurons (DIV5) after EX527 treatment (6 hours). Cells were stained with anti-acetyl MeCP2 or anti-MeCP2 antibody. Bar=65 mm
Figure 3. (A) MeCP2 binding to the BDNF exon IV promoter (200 bp upstream from the transcriptional starting site) is increased in SIRT1Δex4/Nestin-Cre hippocampi as revealed by ChIP analysis (n=3 animals per each genotype). Student's t-test. Values represent means ± SD. *p<0.05, **p<0.01, ***p<0.001. (B) Reduced BDNF protein level in SIRT1Δex4 hippocampi. Level of BDNF mRNA in SIRT1 wild type animals was set as control. Student's t-test (n=3) Values represent means ± SD. *p<0.05, **p<0.01, ***p<0.001.
