Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n−6 to 18:3n−6, 18:2n−6 to 20:2n−6, and 18:3n−3 to 20:3n−3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

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Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

Infection and Immunity ◽

10.1128/iai.01141-09 ◽

2010 ◽

Vol 78 (3) ◽

pp. 1058-1065 ◽

Cited By ~ 16

Author(s):

Chen Zhang ◽

Shao-Hung Wang ◽

Chung-Ping Liao ◽

Shoujin Shao ◽

Mark E. Lasbury ◽

...

Keyword(s):

Alveolar Macrophages ◽

Mrna Level ◽

Gene Promoter ◽

Pneumocystis Pneumonia ◽

Luciferase Reporter ◽

Mrna Levels ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

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A detailed and radioisotope-free protocol for electrophoretic mobility shift assay (EMSA)

10.21203/rs.3.pex-939/v1 ◽

2021 ◽

Author(s):

NGUYEN HOAI NGUYEN

Keyword(s):

Transcription Factor ◽

Electrophoretic Mobility Shift Assay ◽

Electrophoretic Mobility ◽

Chromatin Immunoprecipitation ◽

Direct Interaction ◽

Luciferase Reporter ◽

Mobility Shift ◽

Dna Fragment ◽

Mobility Shift Assay

Abstract To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. This protocol is to provide a detailed procedure for a safe EMSA assay (without using any radioisotope).

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Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences

Journal of Bacteriology ◽

10.1128/jb.00809-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4904-4919 ◽

Cited By ~ 10

Author(s):

Lara L. Hause ◽

Kevin S. McIver

Keyword(s):

Binding Site ◽

Streptococcus Pyogenes ◽

Transcriptional Activation ◽

Luciferase Reporter ◽

Mobility Shift ◽

Content Type ◽

Promoter Sequences ◽

Mobility Shift Assay

ABSTRACTThe Mga regulator ofStreptococcus pyogenesdirectly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1binding site was altered and screened for nucleotides important for DNA bindingin vitroand for transcriptional activation using a plasmid-based luciferase reporterin vivo. Following this analysis, 34 nucleotides within the Pemm1binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemmwith directed mutagenesis performed in the M1T1 Mga-regulated PscpAand Psicpromoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.

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Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element

Biochemical Journal ◽

10.1042/bj20041736 ◽

2005 ◽

Vol 387 (3) ◽

pp. 711-717 ◽

Cited By ~ 127

Author(s):

Eric METZEN ◽

Daniel P. STIEHL ◽

Kathrin DOEGE ◽

Jan H. MARXSEN ◽

Thomas HELLWIG-BÜRGEL ◽

...

Keyword(s):

Binding Site ◽

Oxygen Sensor ◽

Cpg Island ◽

Regulatory Element ◽

Prolyl Hydroxylase ◽

Start Codon ◽

Luciferase Reporter ◽

Mobility Shift ◽

Mobility Shift Assay ◽

Prolyl Hydroxylase Domain

The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1–PHD3) initiate oxygen-dependent degradation of HIF-α-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1α in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5′ of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3′-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.

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Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

Journal of Bacteriology ◽

10.1128/jb.01670-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2496-2504 ◽

Cited By ~ 25

Author(s):

Po-Chi Soo ◽

Yu-Tze Horng ◽

Jun-Rong Wei ◽

Jwu-Ching Shu ◽

Chia-Chen Lu ◽

...

Keyword(s):

Serratia Marcescens ◽

Binding Site ◽

Promoter Region ◽

Transcriptional Start Site ◽

Direct Interaction ◽

Underlying Mechanism ◽

Inhibitory Effect ◽

Start Codon ◽

Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

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Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01634 ◽

2005 ◽

Vol 34 (1) ◽

pp. 177-197 ◽

Cited By ~ 28

Author(s):

A Cote-Vélez ◽

L Pérez-Martínez ◽

M Y Díaz-Gallardo ◽

C Pérez-Monter ◽

A Carreón-Rodríguez ◽

...

Keyword(s):

Luciferase Reporter ◽

Transcriptional Level ◽

Mrna Levels ◽

Mobility Shift ◽

Camp Response Element ◽

Glucocorticoid Response Element ◽

Response Element ◽

Nuclear Extracts ◽

Glucocorticoid Response ◽

Hypothalamic Cells

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

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Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6690-6701.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6690-6701

Author(s):

H Koizumi ◽

M F Horta ◽

B S Youn ◽

K C Fu ◽

B S Kwon ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Site ◽

Protein Interactions ◽

Regulatory Element ◽

Killer Cell ◽

Mobility Shift ◽

Specific Expression ◽

Native Molecular Mass ◽

Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

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Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

Biochemical Journal ◽

10.1042/bj20091500 ◽

2009 ◽

Vol 425 (1) ◽

pp. 235-243 ◽

Cited By ~ 13

Author(s):

Raquel Castro-Prego ◽

Mónica Lamas-Maceiras ◽

Pilar Soengas ◽

Isabel Carneiro ◽

Isabel González-Siso ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Oxygen Availability ◽

Mrna Levels ◽

Mobility Shift ◽

Hypoxic Conditions ◽

Cross Links ◽

Mobility Shift Assay ◽

Transcriptional Adaptation

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1–Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides −557 to −376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

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The role of NeuroD1 in the upregulation of SMYD3 by HBx.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.183 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 183-183

Author(s):

Junyao Xu ◽

Qingqi Hong ◽

Chuanchao He ◽

Jie Wang

Keyword(s):

Gene Expression ◽

Binding Site ◽

Promoter Region ◽

Gene Transfection ◽

Histone Methyltransferase ◽

Proximal Promoter ◽

Luciferase Reporter ◽

Mobility Shift ◽

Cis Element ◽

E Box

183 Background: SET and MYND Domain-Containing Protein 3 (SMYD3) is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis B virus x protein (HBx) is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Methods: Immunohistochemical staining was used to detect the expression of HBx and SMYD3 in HCC tumor tissues. HBx gene transfection, RNAi, and histone methyltransferase(H3-K4) activity assay were performed to reveal the transcrpitionally activation of HBx on functional SMYD3 gene expression. Chromatin immunoprecipitation (ChIP), Co-immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA) were applied to investigate the underlying mechanism. Dual-luciferase reporter assay was used to search for the HBx responsive cis-element of SMYD3 gene. Results: Immunohistochemistry identified the positive correlation between HBx and SMYD3 expression in 42 HCC tissues. Up-regulation of HBx on SMYD3 expression was validated through experiments involving overexpression or knock-down of HBx in different HCC cell lines. And up-regulated SMYD3 is functionally active as histone methyltransferase. Next we found that HBx transcriptionally regulated SMYD3 gene expression by interacting with RNA polymerase IIand altering its binding site to a proximal promoter region(SD2) from a distant promoter region(SD6) of SMYD3. Truncated and mutant reporter assays revealed that the cis-element mapped in -178~-203bp in SMYD3 promotor is responsive for HBx-transactivation. And this 25bp cis-element contains a E-box 3 unit, which is a binding site for the transcriptional factor Neurogenic differentiation 1(NeuroD1). EMSA and Chip showed that HBx increased NeuroD1 binding to SMYD3 proximal promotor, however transcient expression of antisense NeuroD1 abolished HBx-induced SMYD3 expression. Conclusions: HBx transcriptionally up-regulates SMYD3 and that this process is mediated by NeuroD1 through binding to the E-box 3 site of SMYD3 promotor.

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Heat shock of human synovial and dermal fibroblasts induces delayed up-regulation of collagenase-gene expression

Biochemical Journal ◽

10.1042/bj3080743 ◽

1995 ◽

Vol 308 (3) ◽

pp. 743-747 ◽

Cited By ~ 18

Author(s):

E G Hitraya ◽

J Varga ◽

S A Jimenez

Keyword(s):

Gene Expression ◽

Heat Shock ◽

Binding Site ◽

Transcriptional Activation ◽

Human Fibroblasts ◽

Dermal Fibroblasts ◽

Mrna Levels ◽

Hsp 70 ◽

Mobility Shift ◽

Collagenase Gene

We investigated the effect of heat shock on the expression of the collagenase gene in normal human synovial and dermal fibroblasts. Heat shock (42-44 degrees C for 1 h) caused a marked increase in heat-shock protein 70 (HSP-70) mRNA levels, followed by a delayed increase in collagenase mRNA levels, in both cell types. Pretreatment with cycloheximide had no effect on the heat-shock-induced increase in HSP-70 mRNA expression, but abrogated the induction of collagenase mRNA during the recovery. To study the mechanisms of collagenase-gene induction by heat shock, the transcriptional activity of a collagenase-promoter-driven chloramphenicol acetyltransferase (CAT) reporter gene was examined in transient transfection experiments. Heat shock was followed by a > 2-fold increase in CAT activity driven by a 3.8 kb fragment of the collagenase promoter, or by a construct containing an AP-1 binding site. A mutation in the AP-1 binding site abolished the effect of heat shock. Electrophoretic-mobility-shift assays revealed a marked increase in DNA-binding activity specific for the AP-1 binding site in nuclear extracts prepared from synovial fibroblasts recovering from heat shock. These results indicate that heat shock causes a delayed increase in collagenase-gene expression in human fibroblasts, and suggests that this stimulation involves, at least in part, transcriptional activation through an AP-1 binding site. Heat shock appears to initiate a programme of cellular events resulting in collagenase-gene expression, and therefore may contribute to connective-tissue degradation in disease states.

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