Liver receptor homologue-1 is expressed in the adrenal and can regulate transcription of 11 beta-hydroxylase

Liver receptor homologue-1 (LRH-1, designated NR5A2) is a mammalian homologue of Drosophila fushi tarazu factor (dFTZ-F1) and structurally belongs to the orphan nuclear receptor superfamily. LRH-1 can recognize the DNA sequence 5'-AAGGTCA-3', the canonical recognition motif for steroidogenic factor 1 (SF-1). Herein, we hypothesized that LRH-1 might play a role in the regulation of human adrenal expression of steroidogenic enzymes. To test this hypothesis, LRH-1 expression in human adult and fetal adrenal glands was examined by RT-PCR analysis. The fetal and adult adrenal glands, as well as liver and pancreas, were observed to express LRH-1 mRNA using RT-PCR. The ability of LRH-1 to enhance transcription of the gene encoding human 11 beta- hydroxylase (hCYP11B1) was then examined using the H295R adrenal cell line. LRH-1 co-transfection with hCYP11B1 luciferase promoter constructs caused a 25-fold induction of luciferase activity. Furthermore, co-transfection of a hCYP11B1 reporter construct containing a mutation in the SF-1 binding cis-element abolished the stimulatory effect of both SF-1 and LRH-1. Electrophoretic mobility shift assay (EMSA) demonstrated that LRH-1 could bind to the SF-1 response element. Taken together, our data suggested that LRH-1 is expressed in the adrenal, and can substitute for SF-1 to enhance transcription of genes encoding certain of the steroid-metabolizing enzymes. A role for LRH-1 in the regulation of adrenal or gonadal steroid hormone production should be further studied.

Download Full-text

Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

Microbiology ◽

10.1099/mic.0.074286-0 ◽

2014 ◽

Vol 160 (3) ◽

pp. 623-634 ◽

Cited By ~ 7

Author(s):

Tetsu Shimizu ◽

Akira Nakamura

Keyword(s):

Constitutive Expression ◽

Transcriptional Regulator ◽

Pcr Analysis ◽

Mobility Shift ◽

Gene Encoding ◽

Reverse Transcription Pcr ◽

Dnase I Footprinting ◽

Catabolic Genes ◽

Genes Encoding ◽

Mobility Shift Assay

Five genes encoding enzymes required for l-gluconate catabolism, together with genes encoding components of putative ABC transporters, are located in a cluster in the genome of Paracoccus sp. 43P. A gene encoding a transcriptional regulator in the IclR family, lgnR, is located in front of the cluster in the opposite direction. Reverse transcription PCR analysis indicated that the cluster was transcribed as an operon, termed the lgn operon. Two promoters, P lgnA and P lgnR , are divergently located in the intergenic region, and transcription from these promoters was induced by addition of l-gluconate or d-idonate, a catabolite of l-gluconate. Deletion of lgnR resulted in constitutive expression of lgnA, lgnH and lgnR, indicating that lgnR encodes a repressor protein for the expression of the lgn operon and lgnR itself. Electrophoretic mobility shift assay and DNase I footprinting analyses revealed that recombinant LgnR binds to both P lgnA and P lgnR , indicating that LgnR represses transcription from these promoters by competing with RNA polymerase for binding to these sequences. d-Idonate was identified as a candidate effector molecule for dissociation of LgnR from these promoters. Phylogenetic analysis revealed that LgnR formed a cluster with putative proteins from other genome sequences, which is distinct from those proteins of known regulatory functions, in the IclR family of transcriptional regulators. Additionally, the phylogeny suggests an evolutionary linkage between the l-gluconate catabolic pathway and d-galactonate catabolic pathways distributed in Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria.

Download Full-text

The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.861-868.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 861-868

Author(s):

T E Wilson ◽

A R Mouw ◽

C A Weaver ◽

J Milbrandt ◽

K L Parker

Keyword(s):

Nuclear Receptor ◽

Transcriptional Activation ◽

Core Promoter ◽

Orphan Nuclear Receptor ◽

Recognition Sequence ◽

Mobility Shift ◽

Adrenocortical Cells ◽

Gene Encoding ◽

Adult Rat ◽

Gel Mobility Shift

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.

Download Full-text

microRNA and mRNA profiles in the amygdala are relevant to fear memory induced by physical or psychological stress

Journal of Neurophysiology ◽

10.1152/jn.00215.2019 ◽

2019 ◽

Vol 122 (3) ◽

pp. 1002-1022 ◽

Cited By ~ 9

Author(s):

Yan Sun ◽

Wei Lu ◽

Kaixin Du ◽

Jin-Hui Wang

Keyword(s):

Psychological Stress ◽

High Throughput Sequencing ◽

Fear Memory ◽

Physical Stress ◽

Luciferase Reporter ◽

Pcr Analysis ◽

Rt Pcr ◽

Genes Encoding ◽

Reporter Assays ◽

Molecular Profiles

Anxiety is presumably driven by fear memory. Molecular profiles in the amygdala of mice with fear memory induced by psychological and physical stresses remain to be elucidated. Fear memory in mice was induced by a paradigm of social defeat. Physical and psychological stresses (PPS) to an intruder were given by attacks from an aggressive resident. Psychological stress (PS) to an observer was given by the witnessing of aggressor attacks. Amygdala tissues from these mice showing fear memory and anxiety vs. tissues from control mice were harvested to analyze mRNA and microRNA profiles by high-throughput sequencing. In the amygdala of intruders and observers with fear memory, the genes encoding 5-HTR1b, 5-HTR2a, DAR2, AChRM3, and IP3R1 are upregulated, whereas genes encoding GPγ11, GPγ13, GPγT2, RasC3, and P450 are downregulated, indicating that these molecules are involved in fear memory induced by physical/psychological stresses. In the comparison of intruders with observers, the upregulation of genes encoding 5-HTR6, GPγ8, P2R7, NFκ2, CREB3/1, and Itgα9 as well as the downregulation of genes encoding DAR5, 5-HTR1a, and HSP1a are involved in fear memory induced by physical stress. The upregulation of genes encoding DAR1, 5-HTR5a and SSR2/3 as well as the downregulation of AdRα1, CREB3/1, GPγ13 and GPγ8 are involved in fear memory induced by psychological stress. Results obtained by sequencing mRNA and microRNA profiles are consistent with results of quantitative RT-PCR analysis and dual-luciferase reporter assays performed for validation. In conclusion, fear memories and anxiety induced by PPS vs. PS are caused by the imbalanced regulation of different synapses and signaling pathways in the amygdala. NEW & NOTEWORTHY The current study identifies the molecular mechanism underlying fear memory and anxiety induced by psychological stress vs. physical stress, in which the imbalanced expression of microRNA-regulated mRNAs relevant to dopaminergic, adrenergic, and serotonergic synapses in the amygdala plays an important role. This result reveals different molecular profiles for psychological and physical stresses.

Download Full-text

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

Journal of Dental Research ◽

10.1177/154405910808700105 ◽

2008 ◽

Vol 87 (1) ◽

pp. 33-38 ◽

Cited By ~ 3

Author(s):

Y. Wittrant ◽

B. Sriniketan Bhandari ◽

H. Abboud ◽

N. Benson ◽

K. Woodruff ◽

...

Keyword(s):

Dna Synthesis ◽

Binding Motif ◽

Rt Pcr ◽

Mobility Shift ◽

Protein Levels ◽

Macrophage Colony Stimulating Factor ◽

Cellular Pathways ◽

Mobility Shift Assay ◽

Macrophage Colony ◽

Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

Download Full-text

Transcriptional Profiling of Methyltransferase Genes during Growth of Methanosarcina mazei on Trimethylamine

Journal of Bacteriology ◽

10.1128/jb.00420-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5108-5115 ◽

Cited By ~ 17

Author(s):

Christian Krätzer ◽

Paul Carini ◽

Raymond Hovey ◽

Uwe Deppenmeier

Keyword(s):

Transcriptional Profiling ◽

Expression Patterns ◽

Pcr Analysis ◽

Exponential Growth Phase ◽

Methane Formation ◽

Rt Pcr ◽

Transcript Levels ◽

Methanosarcina Mazei ◽

Depth Analysis ◽

Genes Encoding

ABSTRACT The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanol-grown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C1 compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization.

Download Full-text

Pituitary homeobox 1 (Pitx1) stimulates rat LHβ gene expression via two functional DNA-regulatory regions

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01754 ◽

2005 ◽

Vol 35 (1) ◽

pp. 145-158 ◽

Cited By ~ 14

Author(s):

Qiaorong Jiang ◽

Kyeong-Hoon Jeong ◽

Cheryl D Horton ◽

Lisa M Halvorson

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Gene Promoter ◽

Orphan Nuclear Receptor ◽

Mobility Shift ◽

Regulatory Regions ◽

Steroidogenic Factor 1 ◽

Reproductive Axis ◽

Functional Dna ◽

Mobility Shift Assay

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH β-subunit gene (LHβ). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHβ gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHβ gene promoter. While investigating the role of Pitx1 in regulating rat LHβ gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions −73 to −52 in the rat LHβ gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5′Pitx1 site as well as this putative 3′Pitx1 region. In transient transfection analysis, mutation of the LHβ-3′Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5′Pitx1 site with further loss following mutation of the 3′Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHβ gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

Download Full-text

Repression of sigK Intervening (skin) Element Gene Expression by the CI-Like Protein SknR and Effect of SknR Depletion on Growth of Bacillus subtilis Cells

Journal of Bacteriology ◽

10.1128/jb.00625-10 ◽

2010 ◽

Vol 192 (23) ◽

pp. 6209-6216 ◽

Cited By ~ 10

Author(s):

Tatsu Kimura ◽

Yukie Amaya ◽

Kazuo Kobayashi ◽

Naotake Ogasawara ◽

Tsutomu Sato

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Direct Interaction ◽

Lethal Effect ◽

Negative Regulator ◽

Growth Defect ◽

Mobility Shift ◽

Gene Encoding ◽

Initiation Of Replication ◽

Mobility Shift Assay

ABSTRACT The Bacillus subtilis phage DNA-like sigK intervening (skin) element (48 kb) is excised from the chromosome by DNA rearrangement, and a composite gene, sigK (spoIIIC and spoIVCB), is created on the chromosome during sporulation. In this study, we first focused on the role of sknR (skin repressor), which has homology with the gene encoding the Xre repressor of defective phage PBSX. The depletion of SknR caused overexpression of the region between yqaF and yqaN (the yqaF-yqaN operon) and a growth defect in B. subtilis. Point mutation analysis and an electrophoretic mobility shift assay (EMSA) suggested that SknR functions as a negative regulator of gene expression in the yqaF-yqaN operon of the skin element through direct interaction with operators of 2-fold symmetry located in the intergenic region between sknR and yqaF. Deletion analysis revealed that the lethal effect of depletion of SknR was related to overexpression of yqaH and yqaM, whose products were previously reported to associate with DnaA and DnaC, respectively. Furthermore, overexpression of either yqaH or yqaM caused cell filamentation and abnormal chromosome segregation, which suggested that overproduction of these proteins inhibits DNA replication. Moreover, overexpression of yqaM inhibited the initiation of replication. Taken together, these data demonstrate that the B. subtilis skin element carries lethal genes, which are induced by the depletion of sknR.

Download Full-text

The fluorene catabolic linear plasmid in Terrabacter sp. strain DBF63 carries the β-ketoadipate pathway genes, pcaRHGBDCFIJ, also found in proteobacteria

Microbiology ◽

10.1099/mic.0.28215-0 ◽

2005 ◽

Vol 151 (11) ◽

pp. 3713-3722 ◽

Cited By ~ 20

Author(s):

Hiroshi Habe ◽

Jin-Sung Chung ◽

Ayako Ishida ◽

Kano Kasuga ◽

Kazuki Ide ◽

...

Keyword(s):

Gene Cluster ◽

Tricarboxylic Acid Cycle ◽

Bacterial Species ◽

Linear Plasmid ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Gene Encoding ◽

Positive Trait ◽

Tricarboxylic Acid Cycle Intermediates

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the β-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding β-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes γ-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited β-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important β-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

Download Full-text

The Gonococcal Fur Regulon: Identification of Additional Genes Involved in Major Catabolic, Recombination, and Secretory Pathways

Journal of Bacteriology ◽

10.1128/jb.184.14.3965-3974.2002 ◽

2002 ◽

Vol 184 (14) ◽

pp. 3965-3974 ◽

Cited By ~ 45

Author(s):

Shite Sebastian ◽

Sarika Agarwal ◽

John R. Murphy ◽

Caroline Attardo Genco

Keyword(s):

Metal Ion ◽

In Silico Analysis ◽

Exchange Chromatography ◽

Surface Proteins ◽

Promoter Regions ◽

Mobility Shift ◽

Gene Encoding ◽

Fur Protein ◽

Mobility Shift Assay

ABSTRACT In this study, we have characterized the in vitro binding of Neisseria gonorrhoeae Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in Escherichia coli HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, a hypothetical gene (Fe-S homolog), and the opa family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, the Fe-S homolog gene, and the opa gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11 opa genes (opaA through -K), we did not detect binding of purified E. coli Fur with 8 of the 11 opa members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the opa family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.

Download Full-text

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate

The Journal of Biochemistry ◽

10.1093/jb/mvz047 ◽

2019 ◽

Vol 166 (4) ◽

pp. 363-373 ◽

Cited By ~ 2

Author(s):

Hiroshi Hamada ◽

Yuta Goto ◽

Jun Arakawa ◽

Erisa Murayama ◽

Yui Ogawa ◽

...

Keyword(s):

Cell Cycle ◽

Promoter Region ◽

Expression Vectors ◽

Reporter Assay ◽

Rt Pcr ◽

Mobility Shift ◽

A Cell ◽

Mobility Shift Assay ◽

E2f Transcription Factors

Abstract The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5′-upstream of the E2F family genes. The motifs are especially rich in the 5′-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5′-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

Download Full-text