Class I Histone Deacetylase-mediated Repression of the Proximal Promoter of the Activity-regulated Cytoskeleton-associated Protein Gene Regulates Its Response to Brain-derived Neurotrophic Factor

Abstract

Background: Activity-regulated cytoskeleton-associated protein (Arc) transcription is activated by BDNF. Results: BDNF activated the Arc proximal promoter, whereas class I histone deacetylases (HDACs) inhibited this activation. Conclusion: Class I HDACs repress BDNF-induced Arc transcription through its serum response element-containing proximal promoter. Significance: Neuronal Arc expression is regulated by chromatin structure, which may lead to long-lasting changes in neuronal functions.

Neuromodulatory Effect of Gαs- or Gαq-Coupled G-Protein-Coupled Receptor on NMDA Receptor Selectively Activates the NMDA Receptor/Ca2+/Calcineurin/cAMP Response Element-Binding Protein-Regulated Transcriptional Coactivator 1 Pathway to Effectively Induce Brain-Derived Neurotrophic Factor Expression in Neurons

“…Purified BAC DNA (10 g) was electroporated into the ES cell line RENKA, derived from C57BL/6N mice (Fukaya et al, 2006) as described previously (Miya et al, 2008). After selection with G418, recombinant ES clones were identified by PCR with the primers BeIX-U (5Ј-CCACAT-GCTGTCCCCGAGAA-3Ј), BeIX-L (5Ј-CGCCTTCATGCAACCG AAGT-3Ј), and LucIX-L (5Ј-CTCGAAGTACTCGGCGTAGGT-3Ј).…”

“…The pSUPER RNAi System (OligoEngine Platform) was used to knockdown the endogenous expression of CRTC1 according to the instructions of the manufacturer. Oligonucleotides were annealed and subcloned into the HindIII/BglII sites of the vector pSUPER, as described previously (Fukuchi et al, 2014). We evaluated the knockdown efficiency using NIH3T3 cells (overexpressing CRTC1) and rat cortical cells (expressing endogenous CRTC1) as described previously (Fukuchi et al, 2014).…”

“…DNA transfection was performed using calcium phosphate/DNA precipitation as described previously (Tabuchi et al, 2002;Fukuchi et al, 2004Fukuchi et al, , 2015. Briefly, calcium phosphate/DNA precipitates were prepared by mixing 100 l of plasmid DNA (6 g, firefly Luc/Renilla Luc at 10:1) in a 250 mM CaCl 2 solution with an equal volume of 2ϫ HEPES-buffered saline (50 mM HEPES-NaOH, pH 7.05, 280 mM NaCl, and 1.5 mM Na 2 HPO 4 ) and then added to each culture dish.…”

“…Total cellular RNA was extracted by the acid guanidine phenol-chloroform method using TRIsure (Bioline), and 1 g of RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen), as described previously (Fukuchi et al, 2004(Fukuchi et al, , 2014(Fukuchi et al, , 2015. Quantitative PCR amplification was performed using the Stratagene Mx3000p Real-Time PCR system and Brilliant SYBR Green QPCR Master Mix (Stratagene), as described previously (Fukuchi et al, 2004(Fukuchi et al, , 2014. The thermal profile for PCR included an initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 1 min.…”

“…pGL4.12-Bdnf-PIV was generated by inserting rat Bdnf promoter IV (Bdnf-PIV; from Ϫ629 to ϩ281; Tabuchi et al, 2002) into the HindIII/XhoI sites of pGL4.12-luc2CP (Promega). The pGL4.12-Bdnf-PI and pGL4.12-c-fos promoter was constructed by inserting rat Bdnf-PI (from Ϫ528 to ϩ138; Tabuchi et al, 2002) and the human c-fos promoter (from Ϫ404 to ϩ41; Tabuchi et al, 1999) into the HindIII site of pGL4.12-luc2CP, respectively. Ca 2ϩ signal-responsive element 1,2,3 (CaRE1,2,3) and CaRE3-mutated Bdnf-PIV (CaRE1-mutated, 5Ј-acATTTCGcG-3Ј; CaRE2-mutated, 5Ј-ATagTAgaAC-3Ј; CaRE3-mutated, 5Ј-TacCGTac-3Ј; lowercase characters denote mutated nucleotides) were generated with the QuikChange Site-Directed Mutagenesis kit (Stratagene).…”