Figure 3.

Epigenetic modification of the GLT-1 promoter of rat cortical astrocytes by VPA and TSA treatment. (a) The scheme depicts the 5′-UTR of GLT-1 and adjacent promoter region. Empty circles represent unmethylated CpG regions, whereas filled circles represent moderately methylated individual CpGs (∼34%), as measured in untreated cortical astrocytes. The genomic sequence of GLT-1 distal promoter region is shown and putative binding sites for myb-like transcriptional regulator (myb TR) and for nuclear factor-1 (NF-1) are depicted. (b) CpG methylation changes were monitored at individual CpG sites (−1978, −1958, and −1929) after drug exposure. Cortical astrocytes were treated for 72 h with 10 mM VPA, 0.2 μM TSA, 10 μM AMI, and 100 μM LTG. After treatment, genomic DNA was isolated, bisulfite-converted, PCR amplified, and submitted to direct sequencing. Bars represent percentage change of CpG methylation (mean±SE, n=3) compared with untreated cells; t-test: ^*p<0.05 vs control, ^**p<0.005 vs control, ^***p<0.0005 vs control. (c) Cortical astrocytes were exposed to 10 mM VPA for 72 h, genomic DNA was isolated, fragmented and immunoprecipitated using an anti-AcH4 antibody or IgG control. Precipitated DNA was subjected to real-time PCR analysis with a GLT-1 promoter-specific set of primers. AcH4 enrichment is depicted as fold-increase±SE (n=2) compared with untreated cells (set to 1). The results are normalized to input; t-test: ^***p<0.0005 vs control.