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Characterization and functional analyses of the human HTR1A gene: 5' regulatory region modulates gene expression in vitro.

ABSTRACT: BACKGROUND:The serotonin neurotransmitter (5-HT) and its receptors have important roles in neuropsychiatric disorders such as schizophrenia. The aim of this study was to investigate the functional sequences of the 5' regulation region of the human HTR1A gene to explore the effects on the expression of the 5-HT1A receptor. METHODS:Fourteen recombinant pGL3-basic vectors containing deletion fragments of the HTR1A gene regulatory region were transfected with HEK-293 and SK-N-SH cells. The relative chemiluminescence intensities of different length fragments were analyzed. The JASPAR software was used for the prediction of transcription factors. RESULTS:In the HEK-293 cells, the relative chemiluminescence intensity of the -?1649?bp to -?1550?bp (ATG?+?1) fragment was significantly different. Two inhibitory activity regions were found in the -?1409?bp to -?1381?bp and?-?1196?bp to -?1124?bp fragments, which might be bound to the GATA or SOX10 transcription factors as predicted by the JASPAR software. In addition, the fragments located from -?1124?bp to -?1064?bp and from -?908?bp to -?722?bp up-regulated protein expression. Only the sequence from -?1550?bp to -?1409?bp demonstrated a difference in luciferase expression in the both cell lines. According to the results of the 5'-UTR truncated vectors, there was a repression region at the distal end of the 5'-UTR, an enhancer region might be present at the proximal end of the transcription start site. CONCLUSIONS:Although the functional sequences of the HTR1A gene regulatory region were confirmed, the regulatory factors and functional components require further investigation.

SUBMITTER: Wu X

PROVIDER: S-EPMC6311061 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:The glucokinase gene GLK1 of the yeast Saccharomyces cerevisiae is transcriptionally regulated in response to the carbon source of the growth medium. Northern-blot analysis shows that the GLK1 gene is expressed at a basal level in the presence of glucose, de-repressed more than 6-fold under conditions of sugar limitation and more than 25-fold under conditions of ethanol induction. lacZ fusions of the GLK1 gene promoter were constructed and a deletion analysis was performed in order to identify the cis-acting regulatory elements of the promoter that controls GLK1 gene expression. First, the expression seemed to be mediated mainly by one GCR1 and three stress-responsive element (STRE) activating elements. Secondly, an ethanol repression autoregulation (ERA)/twelve-fold TA repeat (TAB) repressor element was identified within the promoter region of the GLK1 gene. A specific and differential protein binding to the STRE was observed with extracts from de-repressed and repressed cells. No differential binding to the GCR1 or ERA/TAB elements was observed with extracts from de-repressed and repressed cells, but, in both cases, the binding was competed for by an excess of the unlabelled GLK1(GCR1) and GLK1(ERA) sequence. The transcription factors Msn2 and Msn4, which bind to the GLK1 upstream region through the STRE, contribute to inductive activation. The transcription factor Gcr1, which binds through the GCR1 element, contributes to constitutive activation. In order to achieve the severe glucose repression of GLK1, constitutive repressor factors acting through the ERA/TAB element must counteract constitutive activation generated by Gcr1 binding to the GCR1 element. Full expression of the GLK1 gene is produced by inductive activation of three STRE when Msn2 and Msn4 proteins are translocated to the nucleus by covalent modification. The combinatorial effect of the entire region leads to the regulated transcription of GLK1, i.e., silent in media with glucose and other preferred carbon sources, such as fructose or mannose, and increased levels of expression upon glucose depletion.

Project description:PurposeAbnormal expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor may potentially increase the susceptibility to neuropsychiatric diseases. The purpose of this study was to investigate the functional sequence of the 3'UTR of the human GRIN1 gene, which encodes the GluN1 receptor to determine the effect on the expression of GluN1 receptor.MethodsWe transferred seven recombinant pmirGLO recombinant vectors containing the 3'UTR truncated fragment of the GRIN1 gene into HEK-293, SK-N-SH, and U87 cell lines and compared the relative fluorescence intensity of adjacent length fragments. The TargetScan database was used to predict miRNAs. Then, miRNA mimics/inhibitors were co-transfected into the three cell lines with the 3'UTR of GRIN1 (pmirGLO - GRIN1), to investigate their influence on GRIN1 gene expression.ResultsCompared with the pmirGLo-Basic vector, the relative fluorescence intensity of the complete GRIN1 gene 3'UTR recombinant sequence -27 bp - +1284 bp (the next base of the stop codon is +1) was significantly decreased in all three cell lines. The relative fluorescence intensities were significantly different between -27 bp - +294 bp and -27 bp - +497 bp regions, and between -27 bp - +708 bp and -27 bp - +907 bp regions. According to the prediction of the TargetScan database and analysis, miR-212-5p, miR-324-3p and miR-326 may bind to +295 bp - +497 bp, while miR-491-5p may bind to +798 bp - +907 bp. After co-transfection of miRNA mimic/inhibitor or mimic/inhibitor NC with a recombinant vector in the 3'UTR region of GRIN1 gene, we found that has-miR-491-5p inhibited GRIN1 expression significantly in all three cell lines, while has-miR-326 inhibitor upregulated GRIN1 expression in HEK-293 and U87 cells.ConclusionmiR-491-5p may bind to the 3'UTR of the GRIN1 gene (+799 bp - +805 bp, the next base of the stop codon is +1) and down-regulate gene expression in HEK-293, SK-N-SH, and U87 cell lines, which implicates a potential role of miR-491-5p in central nervous system diseases.

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Characterization and functional analyses of the human HTR1A gene: 5' regulatory region modulates gene expression in vitro.

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