Fig. 4. Differential regulation of BDNF gene expression by DNA methylation and histone deacetylation. The role of DNA methylation in transcriptional activity of BDNF promoters was investigated by treating rat C6 glioma and mouse Neuro2A neuroblastoma cells with 1 mM 5-Aza-deoxycytidine (5AzadC) for 48 hr. The effects of inhibi- tion of histone deacetylation was studied by treating Neuro2A and C6 cells with 300 nM trichostatin A (TSA) for 48 hr. Muscarinic acetyl- choline receptor M4 gene and constitutive hypoxanthine-phosphori- bosyltransferase (HPRT) gene were used as reference genes. of the protein product. Because of the very low expression levels, we failed to map the transcription start site for rodent BDNF exon VII. However, 50 RACE analysis of rodent exon VII homologue in human showed that transcription initiation site for this exon is located at 285 bp upstream of its 30 end (Kazantseva et al., unpublished). These data strongly suggest that, similarly to BDNF exon I, II, IV, and VI mRNAs (Timmusk et al., 1993), novel exon III, V, VII, VIII, and IXA mRNAs are also transcribed from sepa- rate promoters. Differential Regulation of BDNF Expression by DNA Methylation and Histone Deacetylation Provided that methylation of the cytosine residues in the CpG dinucleotides in genome and posttranslational modiﬁcations of histones in the nucleosome establishes epigenetic codes for gene regulation in different tissues, including nervous system (Hsieh and Gage, 2004; Ballas and Mandel, 2005), we investigated the potential role of chromatin structure on transcriptional activity of BDNF promoters. By treating rat C6 glioma cells and mouse Neuro2A neuroblastoma cells with the DNA methyltrans- ferase inhibitor 5AzadC or with the histone deacetylase (HDAC) inhibitor TSA for 48 hr, we examined the role Journal of Neuroscience Research DOI 10.1002/jnr BDNF Gene Structure in Rodents 531 of DNA methylation and histone acetylation, respectively, in the regulation of BDNF gene expression. We observed robust activation of the expression of BDNF exon I and IV as well as novel exon V, VIII, and IXA transcripts in rat C6 glioma cells after 5AzadC treat- ment (Fig. 4). Expression of exons III and VI in C6 cells was moderately induced by inhibition of DNA methyla- tion. Expression of BDNF exon I and exon III transcripts in Neuro2A cells was signiﬁcantly induced following 5AzadC treatment, whereas there was no change in the levels of other BDNF mRNAs (Fig. 4). In Neuro2A cells, TSA treatment failed to relieve repression of any of the BDNF promoters. However, in C6 cells, inhibition of histone deacetylation by TSA increased the levels of BDNF exon III, exon VII, and exon IXA transcripts. Muscarinic acetylcholine receptor gene M4 was used as a reference because its expression has been shown to be regulated by 5AzadC in various cell lines, other than C6 and Neuro2A, in a cell-type-speciﬁc manner (Lunyak et al., 2002; Wood et al., 2003). Our ﬁndings suggest that DNA methylation and histone deacetylation could play a role in silencing of BDNF gene in a promoter- and cell- speciﬁc manner both in C6 and Neuro2A cells. Activity-Dependent Regulation of Rat BDNF Exon-Speciﬁc mRNAs in the Hippocampus by Kainic Acid-Induced Seizures Glutamate analogue kainic acid induces a rise in in- tracellular Ca2+ levels and differential activation of four previously characterized BDNF promoters in the hippo- campus and cerebral cortex of adult rat brain (Timmusk et al., 1993). We examined whether expression of the BDNF mRNAs containing novel 50 exons is regulated by kainic acid 1, 3, 6, 12, and 24 hr after drug administra- tion. The results revealed differential regulation patterns for BDNF transcripts. BDNF exon I and IV transcripts (exons I and III according to Timmusk et al., 1993) have previously been characterized as the most highly induced BDNF mRNAs in response to kainic acid treatment. It was remarkable that in our experiments not only were these BDNF transcripts induced by kainate but also the levels of novel exon V, VII, VIII, and IXA mRNAs were up-regulated peaking at 3–6 hr posttreatment and rapidly down-regulated to basal levels thereafter (Fig. 5). The levels of exon IV transcripts remained elevated at 3–24 hr posttreatment. BDNF transcripts with exon IIA, IIB, and IIC exhibited differential expression proﬁles in response to kainite treatment. The levels of exon IIC transcripts were markedly elevated at 3 hr, peaked at 6 hr, and de- creased at 12–24 hr after kainate treatment. Expression levels of exon 2A and exon 2B transcripts increased mod- erately at 3 hr, dropped at 6 hr, and reached basal levels at 24 hr posttreatment (Fig. 5). In contrast, the expression levels of BDNF exon III and exon VI mRNAs did not change at any time point studied (Fig. 5). These results agree with the previous reports on the transcript-speciﬁc regulation of rat BDNF mRNAs in response to kainate- induced seizures (Timmusk et al., 1993; Sathanoori et al.,