The SIRT1Dex4/Nestin-Cre mice and the SIRT1
+/+Nestin-Cre mice were kindly provided by Dr. L.H. Tsai.
Primary neuronal cultures
Primary cortical neurons were prepared from wild type pups (1-2 postnatal days). Purified cells were plated using Neurobasal A supplemented with B27 (Invitrogen) on poly-D-lysine coated plates. All the experiments were performed at 5 days in vitro culture (DIV).
Chromatin immunoprecipitation (ChIP)
Hippocampi were dissected from SIRT1Dex4
brain and disuccinimidyl glutarate (DSG) was added to a final concentration of 2 mM for crosslinking. After 45 minutes at room temperature, formaldehyde was added to a final concentration of 1% (v/v) and cells incubated for 15 min. After dual crosslinking, glycine was added to a final concentration of 0.1M and incubated for 10 min to quench formaldehyde. Samples were homogenized, resuspended in lysis buffer and sonicated to shear DNA. The whole-cell extract was incubated over-night at 4°C with the appropriate antibody. Protein G/salmon sperm was added and after two hours beads were washed one time in lysis buffer, one time in low salt buffer (150 mM NaCl), one time in high salt buffer (500 mM NaCl), one time in LiCl, and two times in TE buffer. Bound complexes were eluted from the beads with elution buffer (10%SDS, 0.1M NaHCO3
) followed by heating at 65°C overnight (reverse cross-linking). Immunoprecipitated complexes were treated with RNase A, proteinase K, and phenol:chloroform:isoamyl alcohol extraction. Purified DNA samples were normalized and subjected to real-time PCR. Sequence of BDNF primers12
: BDNF exon IV -200 FW: 5’-GGC TTC TGT GTG CGT GAA TTT GC-3’; BDNF exon IV 0 REW: 5’- AAA GTG GGT GGG AGT CCA CGA G-3’
Immunofluorescence and quantification
Cells were fixed using 4% paraformaldehyde in PBS. Blocking was performed using 3% BSA diluted in PBS for 30 minutes at room temperature. Primary antibody used: anti-acetyl MeCP2 (ABE28 Millipore 1: 400 dilution) was incubated overnight. Secondary antibody was conjugated with Alexa-488 goat anti-rabbit (Invitrogen). Nuclei were stained with DRAQ-5 [-(dimethylamino)ethylamino-4,8-dihydroxyanthracene-9,10-dione] (Biostatus Limited). Immunolabeled sections were examined with a Leica confocal microscope SP5 (DMRE, Leica). Controls were always performed by omitting primary antibodies. Intensity of fluorescence is evaluated as pixels/µm2. The Leica SP5 software LAS AF was used for quantification.
In vitro deacetylation assay
Purified MeCP2e1-FLAG was incubated in deacetylation buffer (50 mM Hepes pH=7.9, 150 mM NaCl, 1mM DTT) in the presence of purified recombinant human SIRT1 (Sirtris Pharmaceuticals) plus 5 mM NAD+ (SIGMA), 1 mM trichostatin A (TSA), 50 mM EX527 (Tocris), 10 mM nicotinamide (NAM) for 1 h at 37°C. The reactions were resolved on SDS PAGE and analyzed by Western blotting.
In vitro acetylation assay
GST-MeCP2e1 was expressed in E. coli BL21. Recombinant proteins were lysed in lysis buffer (20 mM Tris-HCl pH=8, 0.3 mM EDTA, 20% Glycerol, 5mM DTT, 0.5 mM PMSF, 1% Triton X-100, 500 mM KCl) and purified by glutathione Sepharose 4B (Amersham). The purified protein was incubated in acetylation buffer (50 mM Tris-HCl pH=8, 50 mM AcetylCoA (SIGMA), 0.1 mM EDTA, 1mM DTT, 10% glycerol) plus 0.1 mg purified recombinant p300 catalytic domain (Active Motif). The reaction was incubated for 1 hr at 37 °C. Reactions were stopped by the addition of 2X Laemmli loading buffer, followed by SDS PAGE and Western blot analysis. Purified GST-MeCP2e1 was incubated in acetylation buffer (50 mM TrisHCl pH=8, 10% glycerol, 100 mM NaCl, 1 mM DTT, 0.2 mM EDTA, 0.2 mm PMSF, 1 mCi of 14CAcetyl-CoA) in the presence of 0.1 mg recombinant p300 (Active Motif). The reaction was stopped after 1 hr, and the samples were resolved by SDS-PAGE. Gels were stained by Coomassie blue, destained, dried and the level of acetyl MeCP2 was detected by autoradiography.
Antibodies and Western blot
Antibody against FLAG (M2) was from SIGMA, the anti-MeCP2 antibody was from Cell Signaling (MeCP2 D4F3 XPTM) and BDNF antibody from Santa Cruz (BDNF N20 sc-546). c-Myc was from Millipore (clone 9E10). Pan-acetyl lysine antibody (Cell Signaling #9441). The polyclonal acetyl-lysine 464 MeCP2e1 was generated by immunizing rabbits with KHL-conjugates of the peptide NH2-AEK(ac)YKHRGEGE (ABE28Millipore). Specificity of the antibody was validated both in vitro and in vivo by performing the appropriate controls, both in our laboratory and by Millipore/Merck. All Western blots were visualized using a chemiluminescence detection kit (Perkin-Elmer). At least three independent experiments were performed. Densitometry analysis of the film was performed using Adobe Photoshop.
Cell culture, cell extracts and immunoprecipitation.
Human embryonic kidney HEK-293 were maintained at 37°C and 5%CO2, in Dulbecco’s modified Eagle’s high glucose (Thermo Scientific) with antibiotics (penicillin and streptomycin) and 10% newborn calf serum (NCS). Cells were washed in phosphate-buffered saline (PBS) and lysed in RIPA buffer [50 mM Tris pH=8, 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 1% NP40, 1X protease inhibitor cocktail (Roche), 1mM DTT, 1 mM trichostatin A (TSA), 10 mM NAM, 10 mM NaF, 1 mM PMSF]. Immunoprecipitation was performed by pre-clearing 500 mg-1mg of whole lysates with protein G-Agarose beads for two hours, and then by incubating with the appropriate amount of antibody or with anti FLAG-M2 Affinity gel at 4°C (SIGMA).
MeCP2e1-FLAG pCMV-TAG4 (STRATAGENE) was a kind gift of Dr. J.M. LaSalle. Mouse MeCP2e1 cDNA was cloned in a 6-myc/pCDNA3 plasmid. The point mutant MeCP2e1K464R was generated using the Agilent’s QuickChange site-directed mutagenesis kit. The truncation mutants of MeCP2 were generated by PCR amplification followed by cloning in 6-myc/pCDNA3 or pCMV-TAG4 (STRATAGENE). Stratagene QuickChange Site-Directed Mutagenesis Kit was used to generate the single point mutant K464R MeCP2e1.
Differences between two means were assessed with Student’s t-test. At least three independent experiments were performed (n=3). *p<0.05 was considered significant, **p<0.01 and ***p<0.001 were considered highly significant.