Transcriptional Regulatory Role Of Neuron-restrictive Silencer Factor On Cocaine- And Amphetamine-regulated Transcript Gene

Stem cells are a class of cells with strong self-renewal and multilineage differentiation potential, they can be induced to differentiate into mature neurons, islet cells under certain conditions in vitro, and has important application value in cell transplantation to treat diabetes, Parkinson's disease, stroke, etc. However, there is a series of problems in the process of stem cells differentiating into neurons and islet cells, including low efficiency of differentiation, immature cell function and so on, mainly because the differentiation mechanism is unclear. Literature survey and preliminary work in our laboratory have shown that neural restrictive silencer factor (neuron-restrictive silencer factor, NRSF; also known as RE-1 silencing transcription factor, REST) plays important roles in the differentiation of stem cells into neurons and islet cells, it involves in regulating insulin, cocaine- and amphetamine-regulated transcript (CART) and several other genes'expression. Based on these work, further research on NRSF target genes'transcriptional regulation will help us to better understand the mechanisms about NRSF in differentiation of stem cells into neurons or pancreatic islet cells and function maintaining of mature cells.The neuron-restrictive silencer factor, also known as the repressor element (RE)-1 silencing transcription factor (REST), is a Krüppel type zinc finger transcription factor, contains a DNA-binding domain, a lysine-enrichment domain, a proline-enrichment domain and two independent inhibitory domains located at the amino and carboxy termini. NRSF is expressed in embryonic stem cells (ESCs), neural stem cells (NSCs) and non-neural cells, and is absent in most, but not all, differentiated neurons. It was identified as a factor that could interact with a specific consensus 21~23 bp cis-element, a neuron-restrictive silencer element (NRSE/RE1), mediate transcriptional regulation of a series of target genes, such as type II voltage-dependent sodium channel (NaCh II), SCG10, Connexin36, and involve in multiple physiological processes such as maintaining pluripotency and self-renewal of ESCs, playing important roles in early embryonic development and regulating the differentiation of stem cells into neuron or islet cells. Further exploration of transcription regulation mechanisms mediate by NRSF on its'target genes, will give us more information about roles of NRSF in differentiation of stem cells into neuron or islet cells and functions maintaining of mature cells.CART peptides are novel neuropeptides widely distributed in the central, peripheral nervous systems and endocrine tissues, including adrenal glands and pancreatic islet. CART peptides are involved in regulating many physiological processes, consist of food intake and the maintenance of body weight, stress response, energy metabolism and neuroprotection. Some studies confirmed that CART paticipate in glucose-reactivity regulation of islet cells. Over the past decade or so, studies on CART peptides have mostly focused on location, processing and functions. Research elucidating the mechanisms that regulate CART peptides transcription, including transcription factors, regulatory element and neuroprotection by CART is limited. Further investigation of the above scientific issue will direct us to clarify the molecular mechanism of biological functions played by CART, and help us more in the development of therapeutic strategies aimed at diabetes, neural system diseases.Our previous study demonstrated that CART is one of the target genes transcriptionally regulated by NRSF. A putative neuron-restrictive silencer element (NRSE) conserved between the rodent and human CART promoter was identified within the CART gene core promoter and we demonstrated that NRSF specifically binds to the NRSE sequence can negatively regulate CART gene expression. Here we indentified another conserved NRSE sequence highly similar to the consensus NRSE sequence within the intron I of CART by bioinformatics analysis. Whether the intron I NRSE motif similarly participates in the transcription regulation of CART and what is the interaction of these two regulatory domains NRSE? The mechanisms that regulate CART gene transcription have been less well characterized except for the cAMP/PKA/cAMP-response element binding protein (CREB) pathway. How the NRSF-NRSE system coordinate with cAMP/PKA/CREB signal pathway system to maintain a dynamic balance in the critical transcription regulation process of CART gene. To present more evidence elucidating the above issue is the goal of this study.The study consists of three parts:Part I: Regulatory role of intron sequence on CART gene transcription By using rVista software, we analyzed the intron sequences of rat, mouse and human that retrieved from the National Center for Biotechnology Information (NCBI) database, and predicted a series of potential regulatoy element, including ZF5, E47, NRSE, AREB6. Given our previous research with regard to NRSE motif within the promoter of CART, we finally determined to choose the intron NRSE motif as the breakthrough point, in order to further clarify the mechanisms of CART gene transcription. To explore the regulation role of intron sequence and the NRSE motif within it on CART gene transcription, we cloned intact intron I or truncated CART intron I without NRSE fragment into 3'-end of CART promoter-luciferase reporter pGL3-Basic-CART32 (P-Luc) to generate pGL3-Basic-CART32-Intron (P-Luc-I), pGL3-Basic-CART32-NRSEnon Intron (P-Luc-tI), and then transiently transfected these luciferase reporters into NTera2/CloneD1 cells, pRL-CMV plasmid was co-transfected to normalize for luciferase activity. The activity of the P-Luc-I reporter was decreased by 57.9% compared to that of P-Luc vector (P<0.05). The activity of P-Luc-tI reporter was increased by 53.8% relative to P-Luc vector (P<0.05). These results indicate CART intron negatively regulate transcription of CART gene through the NRSE motif within it. NT2/CloneD1 cells were then used for chromatin immunoprecipitation assay with anti-NRSF or anti-IgG antibodies to validate the interaction of NRSF protein to the CART intron in vivo. ChIP PCR products were detected with the anti-NRSF antibody but not detected with control anti-IgG antibody.Part II: Exploration of transcriptional regulatory role and mechanisms of Cocaine- and amphetamine-regulated transcript gene by neuron-restrictive silencer factorOn basis of the original work with regard to the NRSE motif within CART promoter and the preliminary research on intron I NRSE in part I of this academic dissertation. This part we try to further explore how NRSF play a role in the regulation of gene transcription through promoter and intron sequences of CART gene. First of all, we performed Eelectrophoretic Mobility Shift Assay (EMSA) using nuclear cell extracts from HeLa cells, and confirmed that the NRSE sequence of human CART promoter and intron I can binds to NRSF protein in vitro. Results of chromatin immunoprecipitation assay (ChIP) suggest that the NRSE sequences of human CART promoter (pNRSE) and intron I (iNRSE) can bind to NRSF protein in vivo and recruit differential repressor complexes to the pNRSE and iNRSE elements depending on the cell types to regulate CART gene transcription. In order to clarify how NRSF play a role in the regulation of gene transcription through promoter and intron sequences of CART gene, in addition to P-Luc, P-Luc-tI, P-Luc-I, we also generated reporter gene constructs containing truncated CART promoter without NRSE named as pGL3-Basic-NRSEnon promoter (tP-Luc) and containing intact intron I or truncated CART intron I without NRSE fragment at the 3'-end named as pGL3-Basic-NRSEnon promoter-Intron (tP-Luc-I), pGL3-Basic-NRSEnon promoter-NRSEnon Intron (tP-Luc-tI). Then we transiently transfected these reporter constructs into HeLa cells. The results showed that the intron region functions in concerted interaction with promoter region to repress CART promoter activity and pNRSE is a stronger repressor than iNRSE in transcription regulation of CART. A similar activity change for these reporters was observed in NRSF overexpressing SK-N-SH cells. To further evaluate that NRSF-mediated repression was due to the contribution of both NRSE sequences in the regulation of the transcriptional activity of CART, on the basis of pGL3-Control and reporter plasmids which contain two tandem copies of CART pNRSE or mutant pNRSE, we constructed another set of reporter constructs containing two tandem copies of iNRSE or mutant iNRSE at the 3'-end. Transfected these luciferase reporters into HeLa and SK-N-SH cells, and pRL-CMV plasmid was co-transfected to normalize for luciferase activity. The results of luciferase activity assay indicate that both NRSE sequences of CART gene act as repressor sites through interaction with NRSF, and both NRSEs in the promoter and the intron ? are required for efficient repression of CART expression by NRSF.Part III: Crosstalk between positive and negative mechanisms of CART gene expressionTo further investigate the relationship between the NRSF-NRSE negative regulatory pathway and the cAMP/PKA/CREB positive regulatory pathway during the CART transcriptional regulation, measurements of luciferase activity were performed and the results indicated that the NRSF-NRSE system caused by high expression of NRSF can effectively repress the activation of the cAMP/PKA/CREB signal pathway.Furthermore, we established the neural oxygen-glucose deprivation (OGD) model and utilized the capacity of CART peptide to attenuate the ischemia/hypoxia-induced cell death. Some studies showed that CART expression was induced in the cerebral cortex after focal cerebral ischemia in the rat in vivo and after oxygen-glucose deprivation (OGD) in cultured cortical neurons. By using this model we try to further explore the role of NRSF on CART gene transcription and its crosstalk with cAMP/PKA/CREB pathway. SK-N-SH cells grown in 12-well plates were subjected to OGD in a Coy Hypoxia Chamber filled with a hypoxic gas mixture (0.3% O2, 95% N2) for 3 h at 37°C, to acquire an in vitro ischemia/hypoxia neural cell death model. Expression changes of NRSF and CART was determined by RT-PCR and western blot after cells were re-incubated in fresh normal feeding medium at 37°C in 5% CO2 and atmospheric air for 24 h or 48 h. Flow cytometry was used to measure the cell apoptosis. We found that the expression levels of NRSF mRNA and protein increased considerably at 24 h after OGD. In contrast, the expression levels of CART mRNA and protein were decreased at 24 h and 48 h after OGD, and moreover OGD resulted in a marked increase in neural cell apoptosis rate. Using this model, we co-transfected reporter vectors P-Luc-I with pcDNA3.1 or pcDNA3.1-NRSF plasmids into SK-N-SH cells. Luciferase reporter assays showed that the OGD-induced upregulation of NRSF was not sufficient to repress the activation of CART gene mediated by Forskolin/cAMP/PKA/CREB pathway. Only when the NRSF was overexpressed at a higher level, it could inhibit the activity of Forskolin. Apoptosis analysis with flow cytometry confirmed that high level of NRSF repress the role of cAMP/PKA/CREB system.In summary, we found that the NRSE motif in the CART intron I plays an important role in the negative regulation of CART transcription through specific binding to NRSF. The NRSE elements of CART promoter and intron thus coordinate to control the efficient repression of CART transcription. High expression of NRSF can repress the positive regulation of CART by cAMP/PKA/CREB pathway. The present study provides new insight into transcriptional mechanisms of the CART gene by NRSF, and might contribute to development of new strategies for therapeutics and differentiation of stem cells into neuron or islet cells.