Genomic organization and identification of promoter regions for the BDNF gene in the pond turtle Trachemys scripta elegans

Abstract

Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I-III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI-III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression.

Fig. 1

Schematic representation of transcription factor binding sites relative to the transcription start site (TSS) in the promoter region of exons I, II, and III of the turtle BDNF gene using in silico analysis. The numbers indicate the position of nucleotides from the TSS. Abbreviations: C/EBP CCAAT-enhancer binding protein, SP1 specificity protein 1, AP1 activator protein 1, CREBP1 CREB binding protein 1, CREB cAMP response element binding protein, CaRE calcium response element, USF upstream stimulatory factors 1/2, BHLHB2 basic helix-loop-helix B2. The arrows denote the location of primers used for the specific detection of immunoprecipitated DNA fragments containing the targeted transcription factor binding sites for the ChIP assay

Fig. 2

Nucleotide sequence of the 5′ flanking region for tBDNF exons I and II. Numbers on the left represent the position of nucleotides relative to the transcription start site of exon I. Exon sequences are shown in bold uppercase letters. Putative TATA box, CAAT elements, CRE, AP1, SP1, C/EBP sites, and E-boxes are underlined and in bold. Asterisk marks the transcription start sites

Fig. 3

Nucleotide sequence of the 5′ flanking region of tBDNF exon III. Numbers on the left represent the position of nucleotides relative to the transcription start site of exon III. Exon sequences are shown in bold uppercase letters. The locations of putative CRE, AP1, SP1, C/EBP sites, and E-boxes are underlined and in bold. Asterisk marks the transcription start site

Fig. 4

a–c Identification of tBDNF promoter regions for exons I–III analyzed by luciferase reporter assay. The size of each of the constructs is illustrated and the numbering is relative to the transcription start site of each exon. Relative luciferase activity is plotted in the histograms and reflects the transcriptional activity of each of the pBDNF constructs for SHSY5Y and NIH3T3 cell lines. The pGL3-control vector, which contains the SV40 promoter and enhancer sequences, was used as a positive control (Control). The pGL3-Basic vector, which lacks promoter and enhancer sequences, was used as a negative control. Values are from three independent experiments and are represented as means±SEM. Asterisks indicate significant differences from pGL3. P values are given in the text

Fig. 5

ChIP assays were performed to verify whether CREB and BHLHB2 associates with CRE and E-box sites, respectively, identified by in silico analysis of tBDNF promoters. CREB and BHLHB2 antibodies were used to immunoprecipitate cross-linked chromatin from naïve turtle brainstem tissue and PCR performed with primers specific for the tBDNF promoter regions. Control samples and inputs were analyzed in parallel. a–cPCR products show that CREB binds the promoter regions of exon I and exon III but not exon II. d–e BHLHB2 binds to the promoter region of exon II but not to II