Chromatin Immunoprecipitation (ChIP) Assays

ChIP assays were used to detect DNMT1 binding to CpG-rich regions of the GAD67 promoter. The Chip procedure was carried out using a ChIP kit (Upstate). Briefly, approximately 10 mg of the hippocampus was used for this procedure. Tissue was incubated at 37°C for 10 minutes with 500 μL of phosphate buffer saline containing 1% formaldehyde and a cocktail of protease inhibitors (Sigma). Tissue was homogenized in 300 μL of SDS lysis buffer (supplied by ChIP kit, Upstate), and the lysate was sonicated for 15 minutes on ice. Immunoprecipitation was performed overnight at 4°C by the addition of 10 μg of ChIP grade DNMT1 (Abcam) to the sonicated solution. An aliquot of the sonicated lysate without antibody was used as input to quantify the total amount of DNA in sample extracts. Protein-free DNA was extracted and used for detection and quantification of CpG-rich regions of GAD67 promoter by quantitative PCR. The primers of CpG-rich GAD67 promoter were decided by the report of Matrisciano et al. (2013) and shown in Table 1. The level of immunoprecipitated GAD67 promoter by the DNMT1 antibody was expressed as a percentage of the input DNA using the following equation: %(DNA − IP/total input) =  2^{[(Ct(10%input) – 3.32) – Ct (DNA-IP)]} × 100%.