Figure 4.
Glial cell line-derived neurotrophic factor (GDNF) promoter-associated histone H3 is hyperacetylated in astrocytes treated with histone deacetylase (HDAC) inhibitors. (a) Schematic representation of the rat GDNF promoter. The locations of GDNF promoter primers are indicated with arrowheads. (b) Enriched cortical astrocyte cultures were treated with 1.2 mM valproic acid (VPA), 1.2 mM sodium butyrate (SB), or 100 nM trichostatin A (TSA) for 5, 24 or 48 h. Chromatin immunoprecipitation (ChIP) assay was performed using an anti-acetyl-histone H3 antibody. The amount of immunoprecipitated (ChIP DNA) and non-immunoprecipitated genome DNA (input DNA) following 24 h treatment was measured by PCR with GDNF Pa, GDNF Pb and GDNF Pc primer sets. PCR products were run on a 2% agarose gel and stained with ethidium bromide. Representative results from gels for each primer set are shown. (c) Real-time PCR was performed with the GDNF Pc primer set to quantify the ChIP DNA and input DNA after 5 h (□), 24 h (■) or 48 h (■) treatment using a relative standard curve method. The values of the ChIP DNA were normalized to the input DNA. Data are expressed as fold-change over the control and are the means±S.E. of three independent experiments. * p<0.05, ** p<0.01, compared to control groups.