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PubMed2005

Intravertebral cleft sign on fat-suppressed contrast-enhanced MR: correlation with cement distribution pattern on percutaneous vertebroplasty.

RATIONALE AND OBJECTIVES: Filling intravertebral clefts during percutaneous vertebroplasty (PVP) is considered to be important for optimal pain control. It is often difficult to detect clefts on non-contrast MR and some fractures show a solid pattern distribution of injected cement without a cleft sign on non-contrast MR. In this study, we evaluated usefulness of fat-suppressed contrast-enhanced MR to predict a solid pattern distribution of injected cement on PVP. MATERIALS AND METHODS: Twenty-six patients with 35 vertebral compression fractures due to osteoporosis were studied. We performed sagittal T1-weighted, T2-weighted and fat-suppressed contrast-enhanced T1-weighted images prior to PVP. First we evaluated the presence of fluid-filled or gas-containing clefts on non-contrast MR. Next we evaluated contrast-enhanced MR of the same vertebrae for the presence of cleft-shaped unenhanced areas within the diffuse enhancement area. We correlated MR findings with cement distribution patterns of injected cement. RESULTS: Based on MR findings, 35 osteoporotic fractures were divided into 3 types. Type 1 (11 fractures, 31%): There were no clefts on non-contrast MR and no unenhanced areas on contrast-enhanced MR; Type 2 (13, 37%): There were no clefts on non-contrast MR but there were unenhanced areas on contrast-enhanced MR; Type 3 (13, 37%): There were clefts on non-contrast MR and unenhanced areas on contrast-enhanced MR. Of 35 osteoporotic fractures, thirteen vertebral fractures (37%) were noted to contain clefts on non-contrast MR, while 24 vertebral fractures (69%) contained unenhanced areas on contrast-enhanced MR. Cement distributed as a solid pattern within clefts or unenhanced areas in all fractures with them. CONCLUSION: Fat-suppressed contrast-enhanced MR is useful to predict a solid pattern distribution of injected cement prior to PVP.

Aged↗

PubMed2006

The SCL 3' enhancer responds to Hedgehog signaling during hemangioblast specification.

OBJECTIVE: The Hedgehog family of intercellular proteins has a crucial role in embryonic development. Recent experimental data suggests that the Hedgehog pathway may play a role in early hematopoiesis and angiogenesis. Stem cell leukemia (SCL), a basic helix-loop-helix (bHLH) transcription factor, is essential for the specification and function of the hemangioblast. SCL expression in early hematopoietic precursors and endothelium is directed by a 3' enhancer. We hypothesized that the SCL 3' enhancer is regulated by Hedgehog signaling during specification of mesoderm towards hemangioblastic fate. MATERIALS AND METHODS: Whole embryos derived from transgenic mouse lines carrying reporter genes under the regulation of SCL 3' enhancer were cultured in the presence of active Hedgehog peptide. Hedgehog transcriptional regulation of SCL 3' enhancer was studied by in vitro and in vivo binding and reporter assays. RESULTS: Hedgehog induced expansion of cells in which the SCL 3' enhancer was transcriptionally activated. A Gli-binding site within the 3' enhancer of SCL was identified and Gli1 was demonstrated to bind and transactivate this enhancer in a sequence-dependent manner. We further demonstrated that the core region of the SCL 3' enhancer is transcriptionally regulated by Hedgehog in-vivo and that the Gli-binding site located in this enhancer is essential for Hedgehog transcriptional regulation in vitro. CONCLUSION: These findings suggest that SCL may be a direct target of Hedgehog signaling during hemangioblast specification.

Animals↗

PubMed2006

The HSS3/4 enhancer of Crlz1-IgJ locus is another target of EBF in the pre-B cell stage of B cell development.

The HSS3/4 enhancer of Crlz1-IgJ locus was first characterized with regard to the activity of HSS1 IgJ promoter in the plasma cells, where both of HSS3/4 enhancer and HSS1 IgJ promoter were found to be opened simultaneously to drive the IgJ gene expression. Unexpectedly, the HSS3/4 enhancer was also found to be opened in the pre-B cells. However, this opening of HSS3/4 enhancer in the pre-B cells could not be related to the IgJ gene expression, because neither the IgJ promoter was opened nor its gene was expressed at the pre-B cell stage of B cell development. Instead, it was postulated that the opened HSS3/4 enhancer might act on some other nearby promoter in pre-B cells, which is now guessed to be the Crlz1 promoter located at 22.5 kb from it. In consistence with this pre-B cell-specific opening of the HSS3/4 enhancer, a pre-B cell-specific in vivo footprint on a sequence similar to the EBF-binding consensus was detected within the enhancer. In this paper, we show that the protein causing the pre-B cell-specific in vivo footprint on a sequence similar to the EBF-binding consensus is truly EBF as judged by EMSA using various oligo-DNA competitors and anti-EBF antibodies. Also, as expected from other previous reports, EBF was shown to be expressed highly in pre-B cells, but very little or not in immature B, mature B and plasma cells using both the cell lines and FACS-sorted normal primary cells. Convincingly, mutations within the EBF site of HSS3/4 enhancer were shown to significantly impair the HSS3/4 enhancer activity in the pre-B cells, but not in the plasma cells.

Animals↗

PubMed2006

Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development.

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.

Animals↗

PubMed2005

Different regions of the mouse nestin enhancer may function differentially in nestin expression in an NSC-like cell line and astrocytes.

Nestin is a characteristic intermediate filament protein expressed in neural stem cells (NSCs). Evidence has shown that it is also found in reactive astrocytes. Previous studies have demonstrated that the second intron of the human and rat nestin genes harbors the central nervous system (CNS) enhancer and the midbrain enhancer, which regulate nestin expression in different regions of CNS during development. In this study, using an NSC-like cell line C17.2 and primarily cultured astrocytes, we show that both C17.2 cells and astrocytes express nestin. To characterize the nestin enhancer in further detail, we cloned the second intron of the mouse nestin gene, which is homologous to the human and rat counterparts as shown by DNA sequencing. Reporter assay indicated that the full-length nestin enhancer was active in both C17.2 cells and astrocytes, consistent with the immunocytochemistry results. However, in C17.2 cells, the enhancer activity was attributed to the highly conserved 3' part, and the 5' part of the enhancer was suppressive to the transcription activation activity of the full-length enhancer. While in astrocytes, both 3' and 5' parts were able to enhance the reporter gene expression. Our data suggested that different regions of the nestin enhancer might have different functions in C17.2 cells and astrocytes: while the 3' region activates transcription in both cell types, the 5' region suppresses in C17.2 cells but activates in astrocytes nestin expression.

Animals↗

PubMed1985

A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.

A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and -524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.

Base Sequence↗

PubMed2001

Enhancer regions of ovine interferon-tau gene that confer PMA response or cell type specific transcription.

Interferon-tau (IFNtau), produced by the trophectoderm of peri-implantation conceptuses in ruminant ungulates, attenuates the uterine production of a luteolytic factor, prostaglandin F(2alpha), resulting in the maintenance of corpus luteum function. However, molecular mechanisms regulating the temporal/spatial expression of IFNtau gene are not clearly understood. The 5'-upstream region of the sheep IFNtau (oIFNtau) gene was examined for its transcriptional regulation in two different cell types; JEG3 cells supported the transactivation of oIFNtau-reporter construct, but HeLa cells did not. In a heterologous SV40 enhancer-oIFNtau promoter or oIFNtau enhancer-SV40 promoter systems, elements required for such cell specific transactivation were localized between -654 and -555 bases, the enhancer, but not the basal promoter region of the oIFNtau gene. In these combinations, high degrees of transactivation were observed in JEG3 cells and the activity was further enhanced by the addition of phorbol 12-myristate 13-acetate (PMA), while those responses were absent in HeLa cells. To identify nucleotide sequences responsible for cell specific expression, transient transfection studies with sequential point mutations in the enhancer elements were executed. Transactivation of oIFNtau enhancer-reporter constructs was primarily regulated by three regions containing AP-1 site, GATA like sequence and site(s) unidentified. In gel mobility shift assays (GMSAs), the AP-1 site located in the enhancer region was recognized by nuclear extracts from both cell types. However, one of the GMSA probes containing GATA-like sequence exhibited different DNA-protein complex patterns in JEG3 and HeLa cells. Observations, in which the same upstream sequence behaved differently due possibly to kinds of nuclear factors available in these cell lines, suggest that such a sequence may be involved in cell specific transactivation of the oIFNtau gene. Furthermore, the same enhancer sequences were also recognized by nuclear extracts from sheep trophoblasts, suggesting that the enhancer sequences between -654 and -555 bases of oIFNtau gene may be functioning in vivo.

Animals↗

PubMed2001

The histone acetyltransferase activity of PCAF cooperates with the brahma/SWI2-related protein BRG-1 in the activation of the enhancer A of the MHC class I promoter.

Major histocompatibility complex (MHC) class I proteins are an essential component of the immune system allowing the organism to protect from viral infections and neoplastic transformation. Expression of the MHC class I genes is regulated by a variety of cis-regulatory promoter elements among which the enhancer A is of particular importance. This enhancer is synergistically activated through AP-1/ATF and NF-kappa B transcription factors. NF-kappa B recruits the histone acetyltransferase (HAT) p300/CREB-binding protein (CBP) to the multiprotein complex bound to the enhancer A. Here we present evidence that acetylation and deacetylation processes are involved in the activation of the enhancer A. The p300/CBP associated factor PCAF, but not p300/CBP, counteracts the repression of the enhancer A mediated by the histone deacetylase HDAC1. Furthermore, overexpression of PCAF results in an increase in the acetylation of histone H4 bound to the enhancer A and HDAC1 counteracts the PCAF-mediated H4 acetylation. The activation function of PCAF requires the p300/CBP binding motif indicating that PCAF might be recruited to the enhancer A through an association with p300/CBP. Moreover, PCAF and the Brahma/SWI2-related protein BRG-1, which is a key factor of the human ATP-dependent chromatin remodelling complex SWI/SNF, synergistically up-regulate the enhancer A. Synergistic activation requires the HAT domain of PCAF. Taken together our data suggest that members of two different groups of chromatin modifying complexes are involved in the activation of the enhancer A of the MHC class I promoter.

Acetylation↗

PubMed1999

Conserved cAMP responsive element and core promoter complex are critical for specificity of the distal T-cell receptor beta chain enhancer for its native promoter.

The Vbeta 8.1 promoter is regulated by T-cell-specific and ubiquitous transcription factors, which bind immediately upstream of and inside the core promoter region. The various Vbeta promoters contain two conserved elements, a cAMP responsive element (CRE) located upstream of the core promoter and a basal initiator flanked by two regulatory motifs. Here we have studied the interplay between the distal enhancer and its native promoter. We show that the remote enhancer acts specifically through its native promoter. Specific enhancer-promoter interplay is mediated through the conserved regions of the Vbeta promoters. Importantly, the conserved CRE serves as a functional recognition element for the enhancer whereas it barely contributes to promoter activity. The other conserved regions surrounding the initiation site are critical for activators that bind at and function through the core promoter region and thereby regulate both promoter and enhancer activity. The enhancer is highly sensitive to E1A-12S, which represses both general and specific enhancer activities. Enhancer activity and promoter-enhancer specificity is, at least in part, mediated by the coactivators CBP/p300.

Adenovirus E1A Proteins↗

PubMed2001

Comparison of unenhanced and contrast-enhanced spiral CT for assessing interval change in patients with colorectal liver metastases.

RATIONALE AND OBJECTIVES: The purpose of this study was to determine whether the interval change in hepatic colorectal metastases as assessed with serial computed tomographic (CT) scans without contrast material enhancement differs from that as assessed using serial, portal dominant phase, contrast-enhanced CT scans. MATERIALS AND METHODS: Unenhanced and contrast-enhanced abdominal CT scans were obtained in 28 patients. Three radiologists separately reviewed serial unenhanced and contrast-enhanced studies to assess the interval change in liver metastases. These radiologists recorded total number of lesions, bidimensional measurements of the largest lesions (as many as three), and overall impressions regarding the interval change (none, worse, or better). RESULTS: Among the 84 judgments (28 patients x 3 radiologists), comparisons of unenhanced and contrast-enhanced CT studies were concordant in 60 assessments (71%). Nineteen (23%) showed mild disagreement. Of these, contrast-enhanced CT studies demonstrated disease stability when unenhanced CT studies demonstrated otherwise in 11 judgments, whereas unenhanced CT studies demonstrated stability when contrast-enhanced CT studies demonstrated otherwise in eight assessments. Furthermore, of the five marked disagreements, two resulted from a conclusion of interval improvement on unenhanced CT studies and a conclusion of interval worsening on contrast-enhanced CT studies, whereas three demonstrated the opposite. Neither set of serial CT studies systematically resulted in under- or overestimation of disease progression (McNemar Q test, P < .25). CONCLUSION: The authors found no consistent pattern to demonstrate that serial unenhanced or contrast-enhanced CT studies resulted in over- or underestimation of disease progression.

Adult↗

PubMed1997

The beta-phaseolin 5' matrix attachment region acts as an enhancer facilitator.

MARs found flanking the beta-phaseolin gene (phas) were tested for insulating activity in an enhancer blocking assay. True insulators should block enhancer dependent expression of a reporter gene when placed between the enhancer and a promoter. Insertion of phas 3' MAR or coding sequences lowered CaMV 35S enhancer driven GUS expression from the phas basal promoter, indicating a distance dependence of the 35S enhancer. 5' MAR or 5' MAR core fragments could not act as independent enhancers when fused to the phas basal promoter, and did not lower expression when inserted in the enhancer blocking assay construct, indicating that they facilitated 35S enhancer expression at a distance when located between the enhancer and the promoter.

Base Sequence↗

PubMed1998

Prognostic significance of CT contrast enhancement within histological subgroups of intracranial glioma.

UNLABELLED: We report the prognostic significance of tumor CT contrast enhancement within histological subgroups in 831 consecutive adult glioma patients of high-grade (n = 516) and low-grade (n = 315) histology. In the present report, a negative prognostic factor is associated with shortened survival. METHODS: Survival analysis including Kaplan-Meier plots, log-rank tests, Cox analysis, and Aalen's linear model as implemented in SPSS and S-PLUS. RESULTS: Sensitivity and specificity of contrast enhancement as a test for high-grade glioma was 0.87 and 0.79, respectively. Enhancement was a strong negative prognostic factor comparable to high-grade histology in the total patient population. Enhancement was also a negative prognostic factor within the subgroups adult high-grade (Grade 3-4), anaplastic (Grade 3), and low-grade (Grade 1-2) gliomas (p < 0.001). The prognostic implications of initial enhancement declined in high-grade patients surviving beyond 36 months. Tumor contrast enhancement or calcifications (in parentheses) were present in 96% (3.6%) of glioblastomas, in 87% (7.4%) of high-grade gliomas, in 56.5% of anaplastic gliomas, and in 21% (16.2%) of low-grade gliomas. Calcification was a positive prognostic factor within the high-grade group of patients (p < 0.0001). CONCLUSION: Enhancement was a major prognostic factor comparable to high-grade histology in this glioma patient population. Enhancement was a negative prognostic factor within each of the adult subgroups high-grade, anaplastic (grade 3), and low-grade gliomas. Enhancement was strongly associated with but not pathognomonic for high-grade histology.

Adult↗

PubMedDate not supplied

Synergism between immunoglobulin enhancers and promoters.

Enhancers are DNA sequences that stimulate transcription from eukaryotic promoters. This stimulatory effect can be exerted over large distances and from a position either 5' or 3' of a promoter. Enhancers have been found in the genomes of many viruses, and in some cellular genes such as those encoding immunoglobulin heavy chain and kappa light chain. An important feature of both viral and cellular enhancers is the ability of each enhancer to stimulate transcription from many promoters other than the one with which it is found associated. However, the question of whether cellular enhancers stimulate their 'own' promoter more efficiently than other promoters has apparently not been investigated. We show here that the kappa light-chain enhancer stimulates a kappa promoter about 20-fold more than it stimulates either the simian virus 40 (SV40) early promoter or a metallothionein (MT) promoter, two promoters that are very sensitive to other enhancers. Similarly, the heavy-chain enhancer stimulates a heavy-chain promoter much more than it stimulates the SV40 and MT promoters. This synergism between immunoglobulin enhancers and promoters might be due to the action of a protein that binds specifically to each of the regulatory elements.

Animals↗

PubMed2001

Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vectors.

The native PSA enhancer and promoter confer prostate-specific expression when inserted into adenovirus vectors capable of efficient in vivo gene delivery, although the transcriptional activity is low. By exploiting properties of the natural PSA control regions, we have improved the activity and specificity of the prostate-specific PSA enhancer for gene therapy and imaging applications. Previous studies have established that androgen receptor (AR) molecules bind cooperatively to AREs in the PSA enhancer core (-4326 to -3935) and act synergistically with AR bound to the proximal promoter to regulate transcriptional output. To exploit the synergistic nature of AR action we generated chimeric enhancer constructs by (1) insertion of four tandem copies of the proximal AREI element; (2) duplication of enhancer core; or (3) removal of intervening sequences (-3744 to -2855) between the enhancer and promoter. By comparing to the baseline construct, PSE, containing the PSA enhancer (-5322 to -2855) fused to the proximal promoter (-541 to +12), the three most efficacious chimeric constructs, PSE-BA (insertion of ARE4), PSE-BC (duplication of core) and PSE-BAC (insertion of core and ARE4), are 7.3-, 18.9-, and 9.4-fold higher, respectively. These chimeric PSA enhancer constructs are highly androgen inducible and retain a high degree of tissue discriminatory capability. Initial biochemical studies reveal that the augmented activity of the chimeric constructs in vivo correlates with their ability to recruit AR and critical co-activators in vitro. The enhanced activity, inducibility and specificity of the chimeric constructs are retained in an adenoviral vector (Ad-PSE-BC-luc). Systemic administration of Ad-PSE-BC-luc into SCID mice harboring the LAPC-9 human prostate cancer xenografts shows that this prostate specific vector retained tissue discriminatory capability compared with a comparable cytomegalovirus (CMV) promoter driven vector.

Adenoviridae↗

PubMed1999

Enhancer and silencer binding proteins involved in the rat cdc2 promoter activation at the G1/S boundary.

BACKGROUND: Expression of the rat cdc2 gene during G1-S phase progression is negatively and positively regulated by the silencer and enhancer elements located upstream of the basal promoter. The silencer and enhancer sequences resemble each other, but the silencer contains extra internal AG residues. RESULTS: The cDNA clones encoding the enhancer binding proteins cdc2E1 and cdc2E2 were isolated by South-Western blotting. cdc2E1 and cdc2E2 comprise 436 and 256 amino acids and have two RNA binding domains which contain an RNP1 octamer and an RNP 2 hexamer. Both cdc2E1 and cdc2E2 bind to the double-stranded and single-stranded silencer and enhancer sequences, but their binding affinity to the enhancer was stronger than that to the silencer. Transfection of quiescent 3Y1 cells with the cdc2 promoter-luciferase constructs, followed by serum stimulation, showed that the promoter activation at the G1-S phase boundary was reduced greatly by base substitutions within the enhancer, but not within the silencer. Gel shift assays with oligonucleotides containing both the silencer and enhancer showed that formation of the large complex was greatly reduced if base-substitutions were introduced into the enhancer, but not within the silencer. The complex was supershifted completely by anti-cdc2E1 antibody and partially by anti-cdc2E2 antibody. CONCLUSION: These results suggest that cdc2E1 and cdc2E2 preferentially form the multimeric complex at the enhancer site after the late G1 phase for activation of the cdc2 promoter.

Amino Acid Sequence↗

PubMed2000

Polarity of transcriptional enhancement revealed by an insulator element.

Transcriptional enhancers for genes transcribed by RNA polymerase II may be localized upstream or downstream of the stimulated promoter in their normal chromosomal context. They stimulate transcription in an orientation-independent manner when assayed on circular plasmids. We describe a transient transformation system to evaluate the orientation preference of transcriptional enhancers in Drosophila. To accomplish this, the gypsy insulator element was used to block bidirectional action of an enhancer on circular plasmids. In this system, as in the chromosome, blocking of enhancer activity requires wild-type levels of the su(Hw) protein. We evaluated the orientation preference for the relatively large (4.4 kb) Adh larval enhancer from Drosophila melanogaster, used in conjunction with a luciferase reporter gene under the control of a minimal Adh promoter. An orientation preference was revealed by insertion of a single copy of the insulator between the enhancer and the promoter. This orientation effect was greatly amplified when the promoter was weakened by removing binding sites for critical transcription factors, consistent with a mechanism of insulator action in which the insulator intercepts signals from the enhancer by competing with the promoter. The orientation preference, as much as 100-fold, is a property of the enhancer itself because it is displayed by gene constructions introduced into the chromosome regardless of the presence of the insulator in a distal location. These findings are most easily reconciled with a facilitated tracking mechanism for enhancer function in a native chromosomal environment.

Animals↗

PubMed1997

PU.1 can participate in an active enhancer complex without its transcriptional activation domain.

The transcription factor PU.1 is necessary for the development of multiple hematopoietic lineages and contributes to the activity of the immunoglobulin kappa 3' enhancer. A variety of proteins bind to the 3' enhancer (PU.1, PIP, ATF1, CREM, c-Fos, c-Jun, and E2A), but the mechanism of 3'-enhancer activity and the proteins necessary for its activity are presently unclear. We show here that PU.1 participates with other transcription factors in forming a higher-order complex with 3'-enhancer DNA sequences. Each protein is necessary for formation of this complex. Individually, transcription factors that bind to the 3' enhancer do not appreciably stimulate transcription in a cell type in which the 3' enhancer is normally silent (NIH 3T3). However, mixture of multiple transcription factors (PU.1, PIP, c-Fos, and c-Jun) can greatly activate the enhancer. PU.1 is necessary for maximal enhancer activity, but mutants of PU.1 that lack the transcriptional activation domain are nearly as efficient at stimulating enhancer activity as the wild-type PU.1 protein. PU.1 apparently can activate transcription by playing an architectural role in interactions with other transcription factors.

DNA-Binding Proteins↗

PubMed1996

Liver-specific enhancer of the glucokinase gene.

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.

Animals↗