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PubMed1996

Characterization of the cyclic adenosine 3',5'-monophosphate response element of the rabbit surfactant protein-A gene: evidence for transactivators distinct from CREB/ATF family members.

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5'-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an approximately 43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREpal as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

Activating Transcription Factor 2↗

PubMed1999

Ternary complex factors Elk-1 and Sap-1a mediate growth hormone-induced transcription of egr-1 (early growth response factor-1) in 3T3-F442A preadipocytes.

In our search for transcription factors induced by GH, we have analyzed immediate early gene activation in a model of GH-dependent differentiation. Here we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors responsible for its transactivation. Binding activity of egr-1 in electrophoretic mobility shift assay (EMSA) increased transiently 1 h after GH stimulation, accompanied by a concomitant increase in egr-1 mRNA. egr-1 induction appeared not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA synthesis. Truncations of the proximal 1 kb of the egr-1 promoter revealed that a 374-bp region (-624 to -250) contributes about 80% of GH inducibility in 3T3-F442A cells and approximately 90% inducibility in CHO-K1 cells. This region contains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. Use of these site-specific mutations in EMSA showed that disruption of either Ets or SRE sites abrogated ternary complex formation at the composite sites. DNA binding of ternary complexes, but not binary complexes, in EMSA was rapidly and transiently increased by GH. EMSA supershifts indicated these ternary complexes contained serum response factor (SRF) and the Ets factors Elk-1 and Sap-1a. Coexpression of Sap-1a and Elk-1 resulted in a marked increase in GH induction of egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of these Ets factors at multiple sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximizes this response.

Adipocytes↗

PubMed2003

Mobility-shift analysis with microfluidics chips.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Computer Simulation↗

PubMed2002

[Identification of AP1 cis-element and transcriptional effect on cytokeratin 13 gene expression].

OBJECTIVE: To identify the type of CTGAATCA from -nt.199 to -nt.192 of the cytokeratin 13(CK13) gene 5' flanking region and determine its transcriptional effect on CK13 gene expression. METHODS: The CAT systems were used to assess the effects of different motifs of CK13 gene 5' flanking region on transcription. The clones of pCAT-enhancer with the total length, -nt.207 to +nt.63 and the same length of -nt.207 to +nt.63, but the T, G of -nt.198, -nt.197 being changed to A, T of the CK13 gene 5' flanking region, were constructed and transferred to HeLa cells with the help of lipofectin. Then work was done to detect the instant CAT expression of different clones and evaluate the effects of CTGAATCA of the 5' flanking region on CK13 gene expression. The type of the cis-element of CTGAATCA was identified with electrophoretic mobility shift assay (EMSA) and competition-EMSA. RESULTS: CTGAATCA in the CK13 gene 5' flanking region is an AP1 cis-element by EMSA and competition-EMSA, it promotes CK13 gene expression. CONCLUSION: CTGAATCA from -nt.199 to nt.192 of the CK13 gene 5' flanking region is an AP1 reaction element, not a cAMP reaction element. It promotes transcriptional activity of CK13 gene 5' flanking region.

5' Flanking Region↗

PubMed1992

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.

Animals↗

PubMedDate not supplied

Cell-type-specific transcription factor interactions with cis-elements present in the mouse LDH/C proximal promoter region.

The major aim of this study was to identify cell-type-specific transcription factors present in somatic and germ cells that interact at cis-elements within the lactate dehydrogenase C (LDH/C) proximal promoter region. Electrophoretic mobility shift assays (EMSAs) were performed using wild-type and mutagenized GC-box LDH/C double-stranded (ds) oligonucleotides to probe nuclear extracts from primary germ cell populations, and an LDH/C nonexpressing cell line (Hela cells). Significant differences were observed in the composition of nuclear transcription factors bound to cis-elements within the LDH/C proximal promoter region in somatic versus germ cells. Using the wild-type LDH/C ds oligonucleotide to probe germ cell nuclear extracts, we consistently observed two DNA-protein complexes, one formed by Sp1 proteins and one formed by another uncharacterized germ cell nuclear transcription factor. Methylation interference analysis identified a unique 5'G residue within the GC-box motif as an integral part of the cis-element recognized by this germ cell nuclear transcription factor. In contrast, at least six major DNA-protein complexes were observed in EMSA studies using Hela cell nuclear extracts probed with the LDH/C wild-type oligonucleotide. In these EMSA studies, supershift analyses with antibodies recognizing the somatic Sp1 protein and the GC-box mutated LDH/C ds oligonucleotide demonstrated that only one of the six complexes formed was due to binding of Sp1 proteins. These in vitro EMSA studies have identified several somatic nuclear factors and at least one other germ cell nuclear transcription factor, which in addition to members of the Sp1 protein family, recognized cis-elements within the LDH/C proximal promoter region. Observations presented in this report strongly suggest that these somatic and germ cell nuclear transcription factors play central roles in modulating transactivation or suppression of the mouse LDH/C subunit gene in the respective cell types.

Amino Acid Sequence↗

PubMed2000

Aging-dependent proteolysis of NF-kappaB in human fibroblasts.

We investigated the NF-kappaB-like factor induced in the late-passage human oral mucosal fibroblasts stimulated with interleukin-1 (IL-1). Compared with the NF-kappaBs of HeLa cells and early-passage fibroblasts, the NF-kappaB-like factor of late-passage (passage 15) fibroblasts migrated faster in the electrophoretic mobility shift assay (EMSA) and behaved like a 70-80 kDa protein in the gel filtration chromatography. Both antibodies against p50 and p65 subunits of NF-kappaB could supershift the small NF-kappaB-like factor of late-passage cells in the EMSAs. A 47-kDa band was detected in late-passage fibroblasts by immunoblotting against p50. The mobility of the trypsin-degraded NF-kappaB of HeLa cells corresponded to that of the small NF-kappaB-like factor of late-passage fibroblasts in the EMSAs. Furthermore, when the nuclear extracts of the IL-1-stimulated HeLa cells were incubated with those of the IL-1-stimulated old fibroblasts, the p65-p50 NF-kappaB band disappeared, leaving behind a small NF-kappaB-like band. This reduction of NF-kappaB was prevented by the addition of a cysteine protease inhibitor leupeptin. These results suggest that the small NF-kappaB-like factor of late-passage fibroblasts is a part of the NF-kappaB truncated by aging-induced protease(s).

Adolescent↗

PubMed1996

Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box.

In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.

Cell Nucleus↗

PubMed2001

Simultaneous immunoblotting analysis with activity gel electrophoresis in a single polyacrylamide gel.

We describe here that a simple diffusion blotting method can couple immunoblotting analysis with another biochemical technique in a single polyacrylamide gel. The efficiency of protein transfer was evaluated by serial dilutions of nephrosin, a metalloproteinase of the astacin family, and by immunodetection. It is estimated that diffusion blotting produces 25-50% of the signal intensity compared to the classical electrophoretic transfer method. However, with diffusion blotting it is possible to generate several replicas from a single gel. In addition, a protein blot can be obtained from a sodium dodecyl sulfate (SDS)-polyacrylamide gel for zymography assay or from a native polyacrylamide gel for electrophoretic mobility shift assay (EMSA). In this regard, a particular signal in zymography or EMSA can be confirmed by simultaneous immunoblotting analysis with a corresponding antiserum. Therefore, diffusion blotting allows a direct comparison of signals between gels and replicas in zymography assay and EMSA. These advantages make diffusion blotting desirable when partial loss of transfer efficiency can be tolerated or be compensated by a more sensitive immunodetection reaction using enhanced chemiluminescence substrates.

Animals↗

PubMed1998

Selectivity of a C-terminal peptide antiserum for different DNA-binding states of the vitamin D receptor.

Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, including those analogous to where it was largely ineffective in shifting the heterodimer. Thus, Ab195 is sensitive to the ligand binding status of the VDR, discriminates heterodimer and homodimer binding interactions, and should provide an additional tool for exploring conformational changes induced in the receptor.

Amino Acid Sequence↗

PubMed1994

Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies.

Because of the growing importance of the Retinoid X Receptors (RXR alpha, beta and gamma) in the retinoid acid signalling pathway, we have prepared polyclonal and monoclonal antibodies directed against these proteins. For this purpose, either the whole mouse RXR alpha protein expressed in E.Coli, or synthetic peptides corresponding to amino acid sequences common to all RXRs or unique to RXR alpha, beta or gamma, were used as antigens. Antibodies recognizing either all three RXR types (alpha, beta and gamma) or specific for each RXR type were obtained. The antibodies were characterized by immunocytochemistry, immunoblotting, immunoprecipitation and electromobility shift assay (EMSA). These antibodies allowed us to detect the presence of RXR alpha proteins in mouse embryos and in mouse embryonal carcinoma cells (F9 and P19 cell lines) by immunoblotting, immunoprecipitation and EMSA whereas RXR beta could be detected only by EMSA and RXR gamma could not be detected by any of these techniques.

Animals↗

PubMed1995

Dissociation of conventional DNA binding and endonuclease activities by an adeno-associated virus Rep78 mutant.

As part of Rep78/68's involvement in adeno-associated virus (AAV) DNA replication, these highly related, AAV encoded proteins bind to the AAV terminal repeat (TR) DNA and endonucleolytically cleave one strand at the terminal resolution site ("trs" nicking activity) of the TR DNA, a site adjacent to the DNA binding site, 21 bps from the nearest GCTC motif. We have constructed a Rep78 mutant, replacing leucine-threonine at amino acids 64-65 with histidine-methionine (64LH65TM). This mutant, expressed as a chimeric protein with maltose binding protein (MBP), displays Mg++ dependent endonuclease activity, but does not bind to the AAV TR as determined in the conventional electrophoretic mobility shift assay (EMSA). It is also found that wild type MBP-Rep78 endonucleolytically nicks at multiple sites in addition to the previously recognized trs site. These data suggest that the nicking activity is independent of conventional DNA binding activity as measured by EMSA and further suggest that a separate form of DNA recognition by Rep78, not measured by the EMSA, is taking place which allows for endonuclease activity.

Amino Acid Sequence↗

PubMed1997

Evaluation of a potential regulatory role for inverted CCAAT boxes in the human topoisomerase II alpha promoter.

Several chemotherapeutic agents act via inhibition of topoisomerase (topo) II activity. Topo II levels appear to correlate with drug sensitivity in vivo. The DNA immediately 5' to the topo II alpha coding region contains five potentially regulatory inverted CCAAT boxes (ICB). Electrophoretic mobility shift assays (EMSA) using oligomers containing the wild type forms of these ICBs show specific DNA-protein binding. Mutations in these ICBs result in loss of protein binding. EMSA competition studies indicate that the four most 3' ICBs (1-4) bind to the same protein(s), while the most 5' ICB (5) binds to a different protein(s). EMSA supershift assays with antibodies to two known CCAAT binding proteins, CBF and CEB/P, indicate that ICBs 1-4 are binding to CBF, but ICB 5 is not bound by either of these proteins.

Antigens, Neoplasm↗

PubMed1999

Electrospray ionization mass spectrometry: a promising new technique in the study of protein/DNA noncovalent complexes.

With the emergence of electrospray ionization mass spectrometry (ESI-MS), mass spectrometry is no longer restricted to the study of small, stable molecules, but has become a viable technique to study large biomolecules as well as noncovalent biomolecular complexes. ESI-MS has been used to study noncovalent interactions involving proteins with metals, ligands, peptides, oligonucleotides, and other proteins. An area where ESI-MS holds significant promise is in the study of protein/DNA interactions. The most common technique employed to study protein/DNA interactions is the electrophoretic gel mobility shift assay (EMSA). Although this technique has and will continue to provide excellent results, ESI-MS has shown the ability to provide detailed results not easily obtainable by EMSA. In this review I will discuss some of the protein/DNA noncovalent interactions that have been measured using ESI-MS, and contrast the results obtained by ESI-MS to those obtained by EMSA.

DNA↗

PubMed1998

Constitutive NF-kappaB levels in human alveolar macrophages from normal volunteers.

Alveolar macrophages regulate the inflammatory and immune responses within the lung through cytokine production. Nuclear factor kappa B (NF-kappaB), a transcription factor, controls the synthesis of cytokines such as interleukin 1beta, tumour necrosis factor alpha, and interleukin 8. In quiescent cells, NF-kappaB is located in the cytosol as a dimer of protein components (p50, p65) bound to an inhibitor (IkappaB). Upon activation, NF-kappaB translocates to the nucleus and binds to DNA. To determine the constitutive level of NF-kappaB activation in non-smoking normal volunteers, immunohistochemical analysis of alveolar macrophages from 29 subjects was performed with antibody directed against the p65 component of NF-kappaB. These results were confirmed in four subjects by electrophoretic mobility shift assay (EMSA). A human monocytic cell line, THP-1 with and without endotoxin stimulation was used as positive and negative controls, respectively. The mean number of positive cells was 4.1%+/-0.8. EMSA performed on whole cell extracts from four normal volunteers demonstrated minimal constitutive binding compared to the positive control. Supershift assay revealed the presence of the p65 dimer. By both immunohistochemistry and EMSA, alveolar macrophages from healthy non-smoking individuals demonstrate minimal NF-kappaB activation. Immunohistochemistry is a sensitive and quantifiable technique requiring only a minimal number of cells, and this technique may be useful in monitoring small changes in NF-kappaB activation in inflammatory diseases of the lung.

Cytokines↗

PubMed2001

Identification of TNF-alpha-sensitive sites in HCMVie1 promoter.

Viral vectors using the human cytomegalovirus immediate-early promoter (HCMVie1 promoter) are potentially efficient tools for gene delivery in vivo to diverse cell types. We previously demonstrated that two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma), inhibited transgene expression from this promoter in skeletal and cardiac myocytes. In this study, electrophoretic mobility shift assays (EMSAs) were performed to identify the TNF-alpha response elements from the HCMVie1 promoter. The results show that TNF-alpha enhances the interaction of nuclear proteins from the C2C12 myocyte line with a single restricted segment of the HCMVie1 promoter. In vitro DNase I footprinting defined precisely the sites of interaction to two elements: nucleotides -1 to 0 and +24 to +36 relative to a transcription initiation cap homologous in the HCMVie1 promoter. These sites contain homologous sequences for cap initiation site (82%) and NFkappaB (62%) sites, respectively. Specificity was further ascertained by competitive EMSAs with wild-type and mutant oligonucleotide probes. Southwestern blotting showed that three proteins (45, 30, and 20 kDa) bound to this TNF-alpha-sensitive element, separately. However, EMSAs failed to prove a role for Yin Yang-1 (YY-1), NFkappaB (p65), or NFkappaB (p50) in binding to these sites. Our results provide evidence for two novel sites in the HCMVie1 promoter that are targets for TNF-alpha enhanced binding of transcription factors.

Antigens, Viral↗

PubMed2004

Mutation at position -132 in the islet amyloid polypeptide ( IAPP) gene promoter enhances basal transcriptional activity through a new CRE-like binding site.

AIMS/HYPOTHESIS: Mutations in the islet amyloid polypeptide ( IAPP) gene may play a potential role in the abnormal regulation or expression of the peptide. The aim of this study was to determine the functional role of the -132 G/A mutation reported in the promoter region of the IAPP gene in a population of Spanish Type 2 diabetic patients. METHODS: We investigated the transcriptional activity using MIN6 cells and luciferase reporter plasmids in several culture conditions. Key regulatory elements of the IAPP promoter region were also analysed by electrophoretic mobility shift assays (EMSA). RESULTS: The mutant construct doubled IAPP transcriptional activity ( p<0.001). Both constructs showed severely reduced promoter activity (four-fold decrease) in the presence of verapamil and diazoxide. In contrast, IAPP promoter activity was doubled after incubation with forskolin or dexamethasone, regardless of the glucose concentrations in the culture media. EMSA revealed that the -132 G/A mutation increased the binding affinity through two DNA-protein complexes. In addition, a cAMP-responsive element binding protein (CREB) was identified by super-shift EMSA. CONCLUSIONS/INTERPRETATION: Our studies show that the wild-type and the mutant constructs are regulated in a similar pattern under all conditions, strongly indicating that the -132 G/A mutation increases basal but not inducible transcription. These results may be explained by new binding to the mutant region through CREB and other transcription factors not yet identified.

Amino Acid Substitution↗

PubMed2006

Prolactin regulatory element binding protein as a potential transcriptional factor for the insulin gene in response to glucose stimulation.

AIMS/HYPOTHESIS: Prolactin regulatory element binding (PREB) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas; however its role in the pancreas is not known. We therefore examined the role of PREB in insulin gene expression. MATERIALS AND METHODS: To analyse the effects of PREB on insulin gene transcription, we employed the luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA). In cells expressing or knocked down for PREB, insulin expression and secretion were determined. RESULTS: PREB was located mainly in nuclei of rat pancreatic beta cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognised by PREB antiserum. This nuclear extract also showed insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter, mediated the transcriptional effect of PREB. In addition, synthesised PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Cells expressing or knocked down for PREB exhibited increased or decreased insulin expression, respectively. CONCLUSIONS/INTERPRETATION: These results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.

Animals↗