ChIP assays were performed using a ChIP Assay Kit (EMD Millipore, Billerica, MA) following the instructions from the manufacturer. Micropunches of PVN and ARC were prepared from the hypothalamus starting from Bregma −0.8–−2.12 and −2.12–−4.52 mm, respectively. The punches from four animals were pooled to run a single immunoprecipitation. Proteins were cross-linked in DMEM plus 1% formaldehyde at 37°C. Protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN,) was added to the lysis buffer and ChIP dilution buffer. Crosslinked DNA was sheared to approximately 300–700 base pairs with five sets of 5-s pulses using a Misonix Sonicator 4000 (Fisher scientific, Pittsburgh, PA), 5 mm tip and set to 50% maximum power. Sonicated cell supernatant was diluted 10-fold in ChIP dilution buffer. A sample was removed after dilution and called the “input” sample. The remaining diluted cell supernatant sample was pre-cleared with Salmon Sperm DNA/Protein A agarose 50% slurry. Primary MeCP2 antibody (1∶1000) (EMD Millipore, Billerica, MA) was added to the remaining precleared supernatant and incubated overnight. Equal concentration of the normal Rabbit IgG (Cell Signaling Technology, Danvers, MA,) was employed as
