Article Figures & Data
Figures
- Fig. 1.
Transcriptional control of GluR2 expression.A, Model of transcriptional repression mediated by the transcriptional repressor REST, which binds the RE1 element. Both N-terminal (Huang et al., 1999) and C-terminal (Ballas et al., 2001) repressor domains recruit histone deacetylase complexes (HDAC), which contain the Siu3 or CoREST corepressors. B, Nissl-stained sections through the hippocampus 6 and 24 hr after pilocarpine treatment, showing thinning of the pyramidal cell layers at 24 but not 6 hr. Dashed triangle, The region of rat hippocampal slices that was microdissected for assays. con, Control.C, Downregulation of GluR2 mRNA in CA3 after pilocarpine. GluR2 and GAPDH mRNA levels were measured by RNase protection assay at 3, 16, and 24 hr after pilocarpine treatment. GluR2 PCR band signals were normalized to GAPDH. Data are presented as mean ± SEM of percentage to their controls (n= 3–4; *p < 0.05 from 0 hr control).
- Fig. 2.
Reduction of histone H4 acetylation over GluR2 promoter after status epilepticus. A, Schematic of the ChIP procedure. IP, Immunoprecipitation.B, Genomic DNA was collected from the hippocampal CA3 region at 3, 8, 24, and 48 hr after injection of pilocarpine (pilo) and immunoprecipitated with anti-acetyl histone H3 (AcH3) or acetyl histone H4 (AcH4) antibodies (Ab) as indicated. Immunoprecipitated GluR2 genomic DNA fragments were detected by PCR. The graph shows the relative intensities of GluR2 PCR bands in acetyl histone H4 lanes. GluR2 band intensities at each time point were first normalized to their DNA inputs and then expressed as a percentage of control at the 0 time point. Input DNA levels were measured on ethidium bromide agarose plates as described in Materials and Methods. Immunoprecipitations performed without a primary antibody resulted in no PCR bands. Data are presented as mean ± SEM of percentage to the untreated or sham-treated control tissues (n = 3–8; *p < 0.05 by ANOVA and post hocDunnett's test).
- Fig. 3.
BDNF upregulation and hyperacetylation of upstream BDNF promoters after status epilepticus. A, The location of the four BDNF promoters (P1–P4) relative to each other is shown. Each BDNF transcript consists of exon 5 and one of the 5′ alternative exons derived from their corresponding promoters. Promoters P1 and P2 are separated by only 563 bp, and P3 and P4 are separated by only 802 bp, whereas promoters P2 and P3 are separated by ∼15 kb.B, Total RNA was isolated from the CA3 region of rats that had experienced 3 hr of pilocarpine (pilo) treatment or from sham controls, as indicated. BDNF exons I, II, III, and IV were amplified by reverse transcription-PCR with 26 cycles. GAPDH mRNA was also amplified as a control. C1, Hippocampal CA3 regions were collected 3 hr after pilocarpine treatment or from sham controls. DNA representing BDNF promoters P1–P4 was amplified by PCR from genomic DNA fragments that had been precipitated by anti-acetyl H4 (AcH4) antibodies (Ab). C2, The level of BDNF P2 promoter associated with acetyl histone H4 was increased and that of the P4 promoter was decreased after status epilepticus (n= 5; *p < 0.05 from untreated or sham-treated controls; ANOVA and post hoc Newman–Keuls test). Thedashed line indicates 100% level.
- Fig. 4.
Trichostatin A prevents SE-induced histone deacetylation at the GluR2 promoter in the CA3 region. Rats cannulated in the lateral ventricle were injected with 1 mg of TSA in 5 ml 1 hr before pilocarpine injection. ChIP samples were prepared from the hippocampal CA3 region 3 hr after pilocarpine treatment.A, Precipitated GluR2 genomic DNA fragments were detected by PCR. AcH4, Acetyl histone H4;pilo, pilocarpine. B, After normalization of the PCR band signals in the acetyl histone H4 lanesto their DNA input levels, data are presented as mean ± SEM of percentage to the untreated or sham-treated controls (n = 14; *p < 0.05). All data sets passed the Kolmogorov–Smirnov test for normal distribution. Analysis of the same data by the nonparametric Wilcoxon signed rank test confirmed that only the pilocarpine condition was different (p < 0.05) from control.
- Fig. 5.
TSA reverses SE-induced histone deacetylation at the GluR2 promoter in the CA3 region. Brain slices prepared from rats after 3 hr of status epilepticus induced by pilocarpine were incubated in ACSF with or without 300 nm TSA for an additional 3 hr.A, Immunoprecipitated GluR2 genomic DNA fragments were detected by PCR. AcH4, Acetyl histone H4;Pilo, pilocarpine. B, After normalization of the PCR band signals in the H4 lanes to their inputs, data are presented as mean ± SEM of percentage to their untreated or sham-treated controls (n = 4; *p < 0.05).
- Fig. 6.
Trichostatin A reverses pilocarpine-induced downregulation of GluR2 mRNA. Brain slices were prepared from rats that had experienced 3 hr of pilocarpine-induced status epilepticus and incubated in ACSF with or without 300 nm TSA for 7 hr. Total RNA was then prepared from CA3 regions. A, GluR2 and GAPDH mRNA levels were measured by RNase protection.B, GluR2 mRNA normalized to GAPDH is shown for the four conditions. Data are presented as mean ± SEM of percentage to their untreated or sham-treated controls (n = 3; *p < 0.05).
