Regulatory role of neuron‐restrictive silencing factor in the specific expression of cocaine‐ and amphetamine‐regulated transcript gene

Abstract

1These authors contributed equally to this work.Abbreviations used: CART, cocaine-and amphetamine-regulated transcript; ChIP, chromatin immunoprecipitation assay; FBS, fetal bovine serum; HDAC, histone deacetylase; INS-1, insulin-secreting cells; NRSE, neuron-restrictive silencer element; NRSF, neuron-restrictive silencer factor; PC12, pheochromocytoma cells; REST, repressor element-1 silencing transcription factor; shNRSF, short hairpin RNA for NRSF; TSA, trichostatin A. AbstractCocaine-and amphetamine-regulated transcript (CART) peptide is an endogenous peptide which is widely expressed in the CNS and PNS as well as in endocrine cells. Despite the functional knowledge about CART, the mechanisms that regulate CART gene transcription are poorly characterized. Here, we showed that neuron-restrictive silencer factor (NRSF) functions as a negative regulator of CART gene expression in neuroendocrine cells. A putative neuron-restrictive silencer element (NRSE) conserved between the rodent and human CART promoter was identified and demonstrated to bind to NRSF in sequence-specific manner by the electrophoretic mobility shift and chromatin immunoprecipitation assays. Ectopic expression of NRSF in pheochromocytoma cells (PC12) and insulin-secreting cells (INS-1) induced a marked reduction in the level of CART mRNA and the activity of CART promoter or NRSE reporter. The CART promoter showed very low activity in endogenous NRSF-expressing HeLa cells. When expression of NRSF was down-regulated in HeLa cells using a RNA interfering technique, the transcriptional activity of the CART promoter or a NRSE reporter was significantly increased. Taken together, our data suggested that CART gene expression in neuroendocrine cells is strictly controlled by NRSF, via a mechanism dependent upon the CART NRSE.

Neuron-restrictive Silencer Factor (NRSF) Represses Cocaine- and Amphetamine-regulated Transcript (CART) Transcription and Antagonizes cAMP-response Element-binding Protein Signaling through a Dual NRSE Mechanism

“…By contrast, little is known about the repression mechanism. This study together, with our previous findings (25), identified a dual NRSE mechanism for CART repression. As far as we know, this is the first evidence to systematically reveal the repression mechanism of CART transcription.…”

“…The cloned reporter gene vector containing the proximal promoter region of the CART gene (Ϫ700 to ϩ39 bp; P-Luc) was a kind gift from Dr. Peter Morgan (19). The reporter gene construct containing the truncated CART promoter (Ϫ700 to ϩ12 bp) without the NRSE sequence (tP-Luc) was described previously (25). The CART intron I region fragment was generated by PCR amplification using genomic DNA from human mesenchymal stem cells as the template, and primers were as follows: sense, 5Ј-GGATCCTGCTGATGCT-ACCTCTG-3Ј; antisense, 5Ј-GTCGACGCCATACTTCTTC-TCATAG-3Ј.…”

“…The pGL3-control plasmid with a supplementary NdeI site was a kind gift from Dr. Xiaofei Zheng. Reporter gene constructs contain two tandem copies of pNRSE (pNRSE-Luc) and site-directed mutant pNRSE (pNRSE mut -Luc) were also described previously (25). Here we similarly synthesized two tandem copies of iNRSE, sense, 5Ј-CCCCGCGGTCAGTACCTGGGACAGCGTCGCTAAG-TTCCCGCGGTCAGTACCTGGGACAGCGTCGCTAAGTT-A-3Ј; antisense, 5Ј-GATCTAACTTAGCGACGCTGTCCCAG-GTACTGACCGCGGGAACTTAGCGACGCTGTCCCAGGT-ACTGACCGCGGGGGTAC-3Ј; and site-directed mutant iNRSE (iNRSE mut ) sense, (5Ј-CCCCGCGGTCGTTAAATGTTA-CGTCGTCGCTAAGTTCCCGCGGTCGTTAAATGTTACG-TCGTCGCTAAGTTA-3Ј; antisense, 5Ј-GATCTAACTTAGC-GACGACGTAACATTTAACGACCGCGGGAACTTAGCGA-CGACGTAACATTTAACGACCGCGGGGGTAC-3Ј.…”

“…Indeed, we showed that NRSF binds to a typical NRSE site in the promoter of CART gene (pNRSE) (25). Depletion of NRSF in HeLa cells mediated by RNA interference (RNAi) results in a significant increase of CART expression and its promoter activity (25).…”

“…Indeed, we showed that NRSF binds to a typical NRSE site in the promoter of CART gene (pNRSE) (25). Depletion of NRSF in HeLa cells mediated by RNA interference (RNAi) results in a significant increase of CART expression and its promoter activity (25). Notably, the repression mechanism by NRSF often involves the recruitment of co-repressor complexes, such as histone deacetylases (HDACs), mSin3, CoREST (co-repressor of REST) (27)(28)(29)(30)(31); and the specificity of co-repressor recruitment depends on cellular context.…”