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Hoxc8 early enhancer of the Indonesian coelacanth, Latimeria menadoensis.

Hoxc8 early enhancer controls the initiation and establishment phase of Hoxc8 expression in the mouse. Comparative studies indicate the presence of Hoxc8 early enhancer sequences in different vertebrate clades including mammals, birds and fish. Previous studies have shown differences between teleost and mammalian Hoxc8 early enhancers with respect to sequence and organization of protein binding elements. This raises the question of when the Hoxc8 early enhancer arose and how it has become modified in different vertebrate lineages. Here, we describe Hoxc8 early enhancer from the Indonesian coelacanth, Latimeria menadoensis. Coelacanths are the only extant lobefinned fish whose genome is tractable to genome analysis. The Latimeria Hoxc8 early enhancer sequence more closely resembles that of the mouse than that of Fugu or zebrafish. When assayed for enhancer activity by reporter gene analysis in transgenic mouse embryos, Latimeria Hoxc8 early enhancer directs expression to the posterior neural tube and mesoderm similar to that of the mouse enhancer. These observations support a close relationship between coelacanths and tetrapods and place the origin of a common Hoxc8 early enhancer sequence within the sarcopterygian lineage. The divergence of teleost (actinopterygii) Hoxc8 early enhancer may reflect a case of relaxed selection or other forms of instability induced by genome duplication events.

Animals↗

MR imaging of the liver: effect of portal hypertension on hepatic parenchymal enhancement using a gadolinium chelate.

The purpose of this study was to prospectively investigate the extent to which reduced portal blood flow in patients with hepatic cirrhosis and portal hypertension affects hepatic parenchymal enhancement during gadolinium-chelate-enhanced dynamic MR imaging. Breath-hold three-dimensional (3D) spoiled gradient-recalled echo (GRE) MR imaging technique obtained after intravenous administration of a gadolinium chelate was used to measure hepatic parenchymal enhancement and time to peak enhancement in 20 patients with hepatic cirrhosis and clinical evidence of portal hypertension (group 1) and in 20 control subjects without portal hypertension (group 2) who were matched for age, sex, and body weight. Mean peak hepatic enhancement values +/- SD and times to peak enhancement +/- SD were determined for both groups of patients. Mean peak enhancement value (+/-SD) was 78.7% +/- 36.2 in group 1 and 91.6% +/- 46.2 in group 2 (not significant). However, in the nine patients in group 1 with splenomegaly, mean peak enhancement value was 61.3% +/- 14.4, whereas it was 93.0% +/- 42.7 in the 11 patients without splenomegaly (P < .05). Mean time to peak enhancement was 84 seconds +/- 23 in group 1 and 54.0 sec +/- 25.0 in group 2 (P < .01). Our results show that mean peak enhancement value of hepatic parenchyma after intravenous administration of a gadolinium chelate is significantly altered for patients with portal hypertension and splenomegaly. In addition, the time to peak enhancement is delayed significantly when portal hypertension is present. Thus, it is possible that the optimal time for imaging the liver during the portal phase must be tailored to the status of the portal system of the patient.

Adult↗

Relationship between contrast enhancement on fluid-attenuated inversion recovery MR sequences and signal intensity on T2-weighted MR images: visual evaluation of brain tumors.

PURPOSE: To investigate the relationship between the degree of contrast enhancement in fluid-attenuated inversion recovery (FLAIR) sequences and tumor signal intensity on T2-weighted images. MATERIALS AND METHODS: A total of 96 patients suspected of having brain tumors were examined by MR imaging, and whenever a brain tumor with an enhancing part larger than the slice thickness was demonstrated on postcontrast T1-weighted images, postcontrast FLAIR images were additionally acquired. The tumor signal intensity on the T2-weighted images was visually classified as follows: equal or lower compared with normal cerebral cortex (group 1), higher than normal cortex (group 2), and as high as cerebrospinal fluid (CSF) (group 3). When a lesion contained several parts with different signal intensities on T2-weighted images, we assessed each part separately. In each group, we visually compared pre- and postcontrast FLAIR images and assessed whether tumor contrast enhancement was present. When contrast enhancement was present on FLAIR sequence, the degree of contrast enhancement in T1-weighted and FLAIR sequences was visually compared. RESULTS: Postcontrast T1-weighted images showed 46 enhancing lesions, including 48 parts, in 31 MR examinations. FLAIR images of the lesion-parts in group 1 (N=18) did not show significant contrast enhancement. In group 2 (N=12), all the parts were enhanced in FLAIR sequences, and three parts were enhanced more clearly in the FLAIR sequences than in the T1-weighted sequences. In group 3 (N=18), all the parts were enhanced equally or more clearly in the FLAIR sequences than in the T1-weighted sequences. CONCLUSION: The signal intensity in FLAIR sequences is largely influenced by both T1 and T2 relaxation time; there is a close relationship between the signal intensity of brain tumors on T2-weighted images and the degree of contrast enhancement on FLAIR sequences. When tumors have higher signal intensity than normal cortex on T2-weighted images, additional postcontrast FLAIR imaging may improve their depiction.

Adenoma↗

Tissue specific phenotypes of polyomavirus A and B enhancer transpositions and duplications: positional and nonpositional effects on replication in lymphoid cells.

We have extended our studies using the mouse lymphoid cell system to determine if polyomavirus (Py) cell type specificity is a consequence of the simple presence of either the A and B enhancers, or if specificity results from specific combinations or arrangements of the individual enhancer elements. Mouse fibroblasts and several T lymphoid cell lines were transfected with wild-type DNA or genomes containing either a deletion of the B enhancer, a transposition of the A and B enhancers relative to their normal positions, or a tandem duplication of either the A or B enhancers. In some cells activation of DNA replication was dependent on the simple presence of the B enhancer and not upon its position, as all genomes containing a B enhancer replicated efficiently. In other cells, deletion or transposition of the B enhancer abolished DNA replication which suggested that activation depended upon the position of this element. The A enhancer core duplication replicated well in lymphoid cell lines, but duplication of the B enhancer did not yield a generally efficient configuration. We have interpreted these results to suggest that individual enhancer components can interact via specific combinational principles to yield cell type specific effects upon Py DNA replication.

Animals↗

Analysis of different contrast enhancement patterns after microbubble-based contrast agent injection in liver hemangiomas with atypical appearance on baseline scan.

BACKGROUND: We describe different possible enhancement patterns in liver hemangiomas with atypical appearance on baseline ultrasound after microbubble-based contrast agent injection. METHODS: From a series of 253 consecutive lesions that were indeterminate on baseline ultrasound and then scanned after injection of air-filled microbubble contrast agent, 65 focal liver lesions were retrospectively selected on the basis of a diagnosis of liver hemangioma on multiphase contrast-enhanced computed tomography (n = 23), magnetic resonance imaging (n = 27), or histology (n = 15). Each lesion was scanned during arterial phase (30 s after microbubble injection) and late phase (5 min after injection). On-site sonologists performed retrospective assessment of contrast-enhancement patterns by consensus. RESULTS: Centripetal fill-in preceded (n = 50) or not preceded (n = 3) by peripheral nodular/rim-like enhancement was the prevalently observed contrast-enhancement pattern, equivalent to the typical enhancement pattern of liver hemangiomas on contrast-enhanced computed tomography or magnetic resonance imaging. In the remaining lesions, additional enhancement patterns (diffuse contrast enhancement with rapid fill-in and a late hyper-isoechoic appearance, n = 6; peripheral nodular enhancement with a late hypoechoic appearance, n = 3; or persistent heterogeneous and hyperechoic appearance, n = 3) were observed. CONCLUSION: Different contrast-enhancement patterns are possible in atypical liver hemangiomas after microbubble injection. Typical centripetal fill-in is the prevalent pattern and its evidence allows diagnosis.

Adult↗

[Ethical aspects of pharmacological cognition enhancement and the use of psychostimulants by children and young persons].

DEFINITION OF THE PROBLEM: Pharmacological cognition enhancement aims at an improvement of cognitive activity and performance in healthy people by means of appropriate drugs. Ethical implications of this kind of cognition enhancement stand in need of reflection. ARGUMENTS: For a number of reasons, the distinction between treatment and enhancement is fuzzy with regard to Attention Deficit Hyperactivity Disorder (ADHD). In consideration of the growing number methylphenidate prescriptions, one question addressed in this article is whether or not psychostimulants are used not only for therapy but also for cognitive enhancement by children and young people. The possibility of a "grey zone" between treatment and enhancement seems to open the field for medicalization of social and pedagogical problems as well as for "hidden enhancement." In clinical practice, the use of stimulants is associated with certain ethical problems concerning diagnosis, treatment and prevention of ADHD. Some of these problems are associated with the possibility of cognition enhancement. In order to evaluate ethical problems of pharmacological cognition enhancement, short-term and long-term consequences of stimulant use need to be taken into account. This refers to the level of transmitter balance in the learning process, to the level of individual learning strategies as well as to the level of interaction. This raises the question (1) of how well adapted the means of enhancement are with regard to the end of a comprehensive education and socialization, and (2) whether there are justifiable limits to the standardization of behavior and knowledge. (3) Moreover, stipulating an autonomous decision as a minimum prerequisite for legitimate cognition enhancement seems inadequate in the case of children and young persons. CONCLUSION: Considering the evidence and the many open questions associated with pharmacological cognition enhancement for children and young persons, it is concluded that it is indeed a morally problematic technique.

Adolescent↗

Detection of enhancer repeats in the long terminal repeats of feline leukemia viruses from cats with spontaneous neoplastic and nonneoplastic diseases.

Enhancer duplication in the long terminal repeat of feline leukemia virus (FeLV) was examined in primary cells from naturally FeLV-infected cats with various neoplastic and nonneoplastic diseases using the polymerase chain reaction. In all cases, a 170-bp band, corresponding to a standard exogenous FeLV with one copy of enhancer, was detected. Repeated enhancer sequences were found in all 8 cases of thymic-form lymphosarcoma, in some cases of lymphosarcoma of other forms (3/8) and myeloid tumors (2/3), and in only 1 of 6 cases with nonneoplastic diseases. The copy number of FeLV proviruses with a repeated enhancer seemed higher than that of those with one copy of enhancer in 3 cases of thymic form lymphosarcoma. In 5 cases of thymic form lymphosarcoma and in 1 case of erythroleukemia, coexistent FeLVs with double and triple enhancers of different sizes were found. Of the enhancer elements, only the SV40 core binding site was found in all the enhancer direct repeats of these FeLVs. All the provirus clones with single and duplicated enhancer sequences from a single tumor showed mutations or deletions characteristic to that tumor, indicating that enhancer repeats may arise in individual animals after infection with a single virus clone. The present findings indicate that FeLV with enhancer repeats generated in the cat is associated with the induction of neoplastic diseases in natural conditions.

Animals↗

Computer-aided detection applied to breast MRI: assessment of CAD-generated enhancement and tumor sizes in breast cancers before and after neoadjuvant chemotherapy.

RATIONALE AND OBJECTIVES: MRI has shown promise in assessing breast cancer patients undergoing neoadjuvant chemotherapy. Computer-aided detection (CAD) for MRI can automatically display tumor enhancement parameters. This study was performed to determine the utility of CAD applied to breast MRI in this patient population. MATERIALS AND METHODS: Fifteen patients with 16 newly diagnosed locally advanced breast cancers were evaluated with MRI before and after neoadjuvant chemotherapy. CAD assessments, including presence or absence of significant enhancement, enhancement profiles, and maximum sizes, were recorded. Pre-chemotherapy and post-chemotherapy enhancement profiles were compared. Sizes were compared to those measured by the radiologist and at final pathology. RESULTS: Prior to chemotherapy, all tumors demonstrated CAD-assessed significant enhancement. Following chemotherapy, 7/16 tumors showed no residual significant enhancement, but all had residual disease at pathology. In those patients with residual enhancement, comparison of the post-chemotherapy to pre-chemotherapy CAD enhancement profiles showed a significant decrease in percentage of washout enhancement (P = 0.0147) in patients with less than 5 mm of residual microscopic disease. Radiologist-measured tumor sizes demonstrated better correlation with sizes at pathology (r = 0.60) than did CAD-generated tumor sizes (r = 0.32). CONCLUSION: CAD may be helpful in assessing changes in MRI enhancement profiles of tumors following chemotherapy. However, CAD-assessed significant enhancement following chemotherapy can be falsely negative for residual malignancy, and CAD tumor sizes are less accurate than those measured by the radiologist in predicting size of residual malignancy. CAD may complement but should not replace the radiologist's assessment of tumors in this patient population.

Adult↗

Efficacy of spectral presaturation of inversion recovery in evaluating delayed myocardial enhancement.

OBJECTIVE: Delayed myocardial enhancement is caused by a variety of cardiovascular diseases. The extent of the enhanced area has been examined by the inversion recovery (IR) method, whereby at the inversion time (TI), normal myocardium shows a low signal intensity. In this sequence, as pericardial fat shows a very high intensity, a delayed enhancement just below the pericardium may be indistinct. To improve the accuracy of delayed myocardial enhancement, we employed the spectral presaturation of inversion recovery (SPIR) method. MATERIALS AND METHODS: Thirty-five patients with symptoms of cardiovascular disease aged between 36 and 80 years old (mean age, 62 years old) were investigated. Thirty were men and five were women. Inversion recovery and SPIR images were obtained 25 min after initial administration of a gadolinium-based contrast material. Each TI, when the signal intensity of the normal myocardium was null, was determined by images obtained at serial different TIs. A radiologist and a cardiologist examined each image by a consensus reading. The extent of myocardial enhancement was described as none, subendocardial, transmural and a random pattern in each case. Images were ranked over three levels and were based on whether myocardial enhancement could be easily detected or whether the contour of the myocardium was visualized precisely. Student's t-test was conducted to compare the quality of two sequences in all patients and in 22 patients who showed delayed myocardial enhancement. RESULTS: The imaging quality in evaluating delayed myocardial enhancement in all patients was superior with IR compared with SPIR, although it was not statistically significant. The imaging quality in the patients with delayed myocardial enhancement was similar between SPIR and IR. SPIR was superior to the IR sequence in two of the four patients who exhibited transmural enhancement. CONCLUSION: SPIR exhibited equivalent image quality to IR in evaluating delayed myocardial enhancement. As it has the potential advantage in patients with rich adipose tissue surrounding the myocardium, it can be an alternative sequence to evaluate myocardial viability.

Adult↗

The archetype enhancer of simian virus 40 DNA is duplicated during virus growth in human cells and rhesus monkey kidney cells but not in green monkey kidney cells.

Archetype SV40, obtained directly from its natural host, is characterized by a single 72-bp enhancer element. In contrast, SV40 grown in cell culture almost invariably exhibits partial or complete duplication of the enhancer region. This distinction has been considered important in studies of human tumor material, since SV40-associated tumor isolates have been described having a single enhancer region, suggesting natural infection as opposed to possible contamination by laboratory strains of virus. However, the behavior of archetypal SV40 in cultured cells has never been methodically studied. In this study we reengineered nonarchetypal 776-SV40 to contain a single 72-bp enhancer region and used this reengineered archetypal DNA to transfect a number of simian and human cell lines. SV40 DNA recovered from these cells was analyzed by restriction endonuclease analysis, PCR, and DNA sequencing. Reengineered archetype SV40 propagated in green monkey TC-7 or BSC-1 kidney cells remained without enhancer region duplication even after extensive serial virus passage. Archetype SV40 grown in all but one of the rhesus or human cell lines initially appeared exclusively archetypal. However, when virus from these cell types was transferred to green monkey cells, variants with partial enhancer duplication appeared after as little as a single passage. These findings suggest (1) that virus with a single 72-bp enhancer may persist in cultured cells of simian and human origin; (2) that variants with partially duplicated enhancer regions may arise within cell lines in quantities below limits of detection; (3) that these variants may enjoy a selective advantage in cell types other than those from which they arose (e.g., green monkey kidney cells); and (4) that certain cell lines may support a selective growth advantage for the variants without supporting their formation. Our data indicate that enhancer duplication may also occur in human as well as rhesus kidney cells. Thus, detection of enhancer region duplication may not, a priori, indicate laboratory contamination, nor does detection of a single 72-bp enhancer exclude the possibility that contamination may have occurred. These findings may be of relevance to studies attempting to detect SV40 DNA in human tumors or other clinical specimens.

Animals↗

Hepatic outflow obstruction: enhancement patterns of the liver on MR angiography.

Our purpose was to present the enhancement patterns of the liver on MR angiography in patients with hepatic outflow obstruction. Twenty-three patients with Budd-Chiari syndrome (4 in acute stage and 19 in chronic stage of the disease) were examined with 3D contrast-enhanced MR angiography. During early and late portal venous phase of MR angiography the pattern of parenchymal enhancement was assessed on source images. The enhancement patterns were evaluated under 4 groups as following: (a) central (b) peripheral (c) patchy and (d) homogeneous enhancement. The morphologic changes in the liver (lobar hypertrophy or atrophy, hepatic surface irregularities) were also recorded. In the acute stage global liver enlargement (75%) with caudate hypertrophy (100%) and central enhancement of the liver (75%) were suggestive findings of the hepatic outflow obstruction. The left lobe hypertrophy (53%) associated with the caudate lobe hypertrophy (72%) and irregular surface (26%) were predominant in the chronic stage of the disease. The enhancement patterns seen in chronic disease were variable and reflected the persistent stasis of the portal blood flow (patchy enhancement in 32% of the patients) or the altered hemodynamics of the liver due to the development of subcapsular collaterals (peripheral enhancement in 21% of the patients). Homogeneous enhancement of the liver in Budd-Chiari syndrome may indicate the chronicity of the outflow obstruction (37%) and shows a more stable hepatic perfusion that occurs after the formation of intra and extrahepatic collateral veins. The morphological and perfusional features on multiphase contrast-enhanced MR angiography are valuable in understanding the effects of the hepatic outflow obstruction on the liver parenchyma.

Acute Disease↗

[Comparison of contrast-enhanced low mechanical index (Low MI) sonography and unenhanced B-mode sonography for the differentiation between synovitis and joint effusion in patients with rheumatoid arthritis].

PURPOSE: To test whether contrast-enhanced low mechanical index (low MI) sonography is superior to non enhanced B-Mode sonography in differentiating synovitis and joint effusion. MATERIAL AND METHODS: In a retrospective study, 22 patients with proven rheumatoid arthritis underwent B-Mode sonography and low-MI sonography of 25 symptomatic joints of the upper and lower limbs. For low-MI sonography, 5 ml Sonovue (Bracco Altana Pharma GmbH, Konstanz) were injected as an intravenous bolus followed by 10 ml of 0.9 % saline solution. Magnetic resonance imaging (MRI) was obtained additionally in 3 joints. With non-enhanced sonography, we diagnosed a synovitis in case of an echogenic and a joint effusion in case of an anechoic mass. With contrast-enhanced sonography, we diagnosed a synovitis in case of enhancement and a joint effusion in the absence of enhancement of the intraarticular mass. RESULTS: In 13 joints, synovitis and joint effusion were differentiated by both non-enhanced and enhanced sonography. In 12 joints, this differentiation was only possible with contrast-enhanced sonography. In 3 patients diagnosed by sonography as having a synovitis, this diagnosis was proven by MRI. CONCLUSION: Contrast-enhanced low-MI sonography is superior to non-enhanced B-Mode sonography in differentiating synovitis and joint effusion.

Adult↗

Evolutionary constraint on Otx2 neuroectoderm enhancers-deep conservation from skate to mouse and unique divergence in teleost.

Otx2 is a paired type homeobox gene that plays essential roles in each step and site of head development in vertebrates. In the mouse, Otx2 expression in the anterior neuroectoderm is regulated primarily by two distinct enhancers: anterior neuroectoderm (AN) and forebrain/midbrain (FM) enhancers at 92 kb and 75 kb 5'of the Otx2 locus, respectively. The AN enhancer has activity in the entire anterior neuroectoderm at headfold and early somite stages, whereas the FM enhancer is subsequently active in the future caudal forebrain and midbrain ectoderm. In tetrapods, both AN and FM enhancers are conserved, whereas the AN region is missing in teleosts, despite overt Otx2 expression in the anterior neuroectoderm. Here, we show that zebrafish and fugu FM regions drive expression not only in the forebrain and midbrain but also in the anterior neuroectoderm at headfold stage. The analysis of coelacanth and skate genomic Otx2 orthologues suggests that the utilization of the two enhancers, AN and FM, is an ancestral condition. In contrast, the AN enhancer has been specifically lost in the teleost lineage with a compensatory establishment of AN activity within the FM enhancer. Furthermore, the AN activity in the fish FM enhancer was established by recruiting upstream factors different from those that direct the tetrapod AN enhancer, yet zebrafish FM enhancer is active in both mouse and zebrafish anterior neuroectoderm at the headfold stage.

Animals↗

Repeated B motifs in the human immunodeficiency virus type I long terminal repeat enhancer region do not exhibit cooperative factor binding.

The enhancer element of the human immunodeficiency virus type I (HIV-I) long terminal repeat (LTR) contains two copies of nearly identical sequences AGGGACTTTCC (3G sequence) and GGGGACTTTCC (4G sequence) that are important in transcriptional regulation. A single copy of the 4G sequence is found in the NF-kappa B site of the immunoglobulin kappa-chain enhancer. Only the 4G motif in the HIV enhancer is bound by cellular proteins in extracts prepared from unstimulated HeLa cells, whereas the 3G and 4G motifs are bound by factors in extracts prepared from HeLa cells treated with phorbol esters [phorbol 12-myristate 13-acetate (PMA)] and lymphoid cells. To determine if this change in binding to the HIV enhancer was due to phosphorylation of a cellular protein, partially purified PMA-treated HeLa nuclear extracts were digested with calf intestinal phosphatase. Phosphatase digestion of nuclear extracts from PMA-treated HeLa cells markedly decreased factor binding to the HIV enhancer. Accordingly, phosphorylation of the DNA binding protein itself, or an inhibitor protein present in the partially purified extract, must mediate binding to the recognition sequence. Binding studies confirmed that each of the enhancer sequences was capable of binding factors independent of the activity of the other site and that the HIV enhancer was occupied by only one factor at any one time. Chloramphenicol acetyltransferase assays using mutants in either one or both HIV enhancer repeats revealed that each site was capable of functioning as a tat-inducible enhancer element in PMA-treated HeLa cells. These results suggest that the 3G and 4G motifs in the HIV enhancer function independently and that duplication in the HIV enhancer augments activity by a mechanism distinct from cooperative binding of NF-kappa B.

Base Sequence↗

Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.

L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.

Animals↗

Interaction of EF-C/RFX-1 with the inverted repeat of viral enhancer regions is required for transactivation.

The hepatitis B virus (HBV) and polyomavirus (Py) enhancer regions contain multiple cis-acting elements that contribute to enhancer activity. The EF-C binding site was previously shown to be an important functional component of each enhancer region. EF-C is a ubiquitous binding activity that interacts with an inverted repeat sequence in the HBV and Py enhancer regions. Although the EF-C binding site is required for optimal enhancer function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. With both the HBV and Py enhancer regions, EF-C stimulates the activity of adjacent enhancer elements in a synergistic manner. EF-C corresponds to RFX-1, a protein that binds to a conserved and functionally important site in major histocompatibility complex (MHC) class II antigen promoter regions. Interestingly, the RFX-1 binding site in MHC class II promoters only contains an EF-C half-site, maintaining one arm of the inverted repeat in an EF-C binding site. We have investigated the binding of purified EF-C and RFX-1 to sites in the Py and HBV enhancer regions that carry mutations that either disrupt one arm of the EF-C inverted repeat, or alter the spacing between the repeats. Our results show that the interaction of EF-C and RFX-1 with an intact inverted repeat is required for functional activity of these viral enhancer regions. Chemical footprinting and modification interference assays show that the interaction of EF-C and RFX-1 with the DRA MHC class II promoter truly represents half-site interaction, and that this binding is unstable. In contrast, the binding of EF-C and RFX-1 to the viral inverted repeats is stable. These results suggest that an additional activity may be required to stabilize EF-C/RFX-1 interaction with the MHC class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-C/RFX-1.

3T3 Cells↗

The Xenopus RNA polymerase I transcription factor, UBF, has a role in transcriptional enhancement distinct from that at the promoter.

Repeated sequence elements found upstream of the ribosomal gene promoter in Xenopus function as RNA polymerase I-specific transcriptional enhancers. Here we describe an in vitro system in which these enhancers function in many respects as in vivo. The principal requirement for enhancer function in vitro is the presence of a high concentration of upstream binding factor (UBF). This system is utilized to demonstrate that enhancers function by increasing the probability of a stable transcription complex forming on the adjacent promoter. Species differences in UBF are utilized to demonstrate that enhancers do not act by recruiting UBF to the promoter, rather UBF performs its own distinct role at the enhancers. UBF function in enhancement differs from that at the promoter, as it is flexible with respect to both the species of UBF and the enhancer element employed. Additionally, we identify a potential role for the mammalian UBF splice variant, UBF2, in enhancer function. We demonstrate that the TATA box binding protein (TBP)-containing component of Xenopus RNA polymerase I transcription, Rib1, can interact with an enhancer-UBF complex. This suggests a model in which enhancers act by recruiting Rib1 to the promoter.

Animals↗

Distance-independent inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding protein of Drosophila.

When the gypsy retrotransposon of Drosophila inserts between an enhancer and promoter it prevents the enhancer from activating transcription. Enhancers are blocked because the protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy. For example, gypsy insertions in an 85 kilobase region between a wing margin-specific enhancer and the promoter in the cut gene cause a cut wing phenotype that is suppressed by su(Hw) mutations. A temperature-sensitive combination of mutant su(Hw) alleles was used to investigate the mechanism by which SUHW blocks the cut wing margin enhancer. By shifting from the nonpermissive to the permissive temperature and vice versa at various stages in development it was found that active SUHW is only required around pupariation when the wing margin enhancer is active to cause a cut wing phenotype. This was true whether gypsy was in the embryonic control region near the promoter, or in the late larval control region near the wing margin enhancer. These results indicate that SUHW must be active only when an enhancer is active to block the enhancer. Furthermore, the observations also indicate that enhancer-blocking by SUHW is reversible and that it occurs soon after binding of active SUHW to gypsy DNA. These results are consistent with models in which SUHW structurally interferes with enhancer-promoter interactions.

Alleles↗