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PubMed2025

Progesterone receptor isoform modulation via enhancer activation regulates progesterone signaling in endometrial stromal cells.

OBJECTIVE: To investigate enhancer-mediated regulation of progesterone receptor (PGR) isoforms, PGR-A and PGR-B, in human endometrial stromal cells, and to determine how isoform modulation shapes the progesterone-responsive transcriptome and cistrome relevant to endometrial function. DESIGN: A clustered regularly interspaced short palindromic repeats-based functional genomic screen was used to identify distal enhancers in telomerase-immortalized human endometrial stromal cells. Subsequent clustered regularly interspaced short palindromic repeats targeting of identified enhancers and the PGR promoter was used to modulate PGR isoform balance and assess functional consequences. SUBJECTS: None. EXPOSURE: Engineered endometrial stromal cells were treated with medroxyprogesterone acetate or vehicle. MAIN OUTCOME MEASURES: PGR isoform expression was assessed by western blot, the progesterone-responsive transcriptome was characterized by bulk ribonucleic acid sequencing, and the PGR cistrome was characterized by Cut&Run. RESULTS: Two distal PGR enhancers were identified in endometrial stromal cells located approximately 60 and 220 kb upstream of the PGR transcription start site. Clustered regularly interspaced short palindromic repeats-based activation of these enhancers upregulated both PGR-A and PGR-B, whereas promoter activation primarily upregulated PGR-B. Bulk ribonucleic acid sequencing revealed that shifting the PGR isoform balance altered the progesterone-regulated transcriptome: PGR-A/B-equivalent cells exhibited proinflammatory gene signatures, whereas PGR-B-dominant cells demonstrated suppression of inflammatory signaling and altered cell cycle programs. The PGR Cut&Run profiling revealed distinct genomic binding patterns associated with each isoform profile. Integration of the PGR cistrome with chromatin interaction maps suggested that these isoforms directly regulate distinct gene subsets involved in inflammation and fibrosis. Mechanistically, estrogen receptor alpha (ESR1) indirectly activated PGR-A expression, potentially through recruitment of Forkhead box protein O1 (FOXO1) at the distal enhancer, suggesting a noncanonical, enhancer-mediated mechanism of PGR regulation. CONCLUSIONS: Distal enhancers regulate the PGR isoform balance and shape the progesterone-responsive transcriptome in human endometrial stromal cells. This enhancer-mediated mechanism expands current models of PGR regulation beyond promoter-level control and may offer potential therapeutic targets to restore normal progesterone responsiveness in conditions marked by PGR isoform imbalance.

Humans↗

PubMed1997

Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro.

The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhanced the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain was sufficient to form a higher-order complex with AML1/Ets-1, but exhibited only partial activity in transcription. We conclude that the T cell-enriched proteins assemble on the enhancer independently of CREB and function synergistically with CREB to activate the TCR alpha enhancer in a chromatin environment.

Base Sequence↗

PubMed2000

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells. METHODS: Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line. We then replaced the luciferase gene with the E. coli cytosine deaminase gene in the subclone that showed the most luciferase activity. The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines. The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared. RESULTS: The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) of 5-FC was similar in all cell lines tested (>10 mM). However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection. CONCLUSIONS: Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease.

Antigens, Surface↗

PubMed2001

PEBP2 and c-myb sites crucial for lambda5 core enhancer activity in pre-B cells.

The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B(lambda5), located 5' of the gene. Here we analyze the lambda5 enhancer core, b(lambda5), which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5 )element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located just upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambda5) element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the lambda5 gene.

Animals↗

PubMed1985

A transcription enhancer in the Herpesvirus saimiri genome.

Herpesvirus saimiri, an oncogenic agent of New World primates, has a linear double-stranded DNA genome of approximately 155 kb. To test its genome for the presence of a transcription enhancer, we have mixed randomly fragmented H. saimiri DNA with non-infectious, linear SV40 DNA lacking the 72-bp repeat enhancer region (the so-called SV40 enhancer trap) and co-transfected this DNA mixture into monkey CV-1 cells. Viable SV40-like viruses were generated by intracellular ligation/repair processes with short H. saimiri DNA fragments. One recombinant, SVHS-2, had integrated a 377-bp enhancer segment from the righthand region of the H. saimiri genome, 7 kb upstream of DNA sequences encoding an immediate-early mRNA. This enhancer sequence is contained within the non-repetitive portions of the viral genome known to be preserved episomally in all lymphoid tumor cell lines. Further recombinant viruses (SVHS-14, SVHS-7, and SVHS-8) essentially contain subsets of the 377-bp insert. Unlike in the previous enhancer trap experiments, where heterologous enhancers were incorporated without any sequence alterations, SVHS-14 and SVHS-7 have suffered short internal deletions of a very similar segment of the H. saimiri insert. This renders the enhancer more active, implying that the deleted segment, while it may have a role in the herpesvirus infection cycle, exerts a negative effect within the isolated enhancer.

Antigens, Viral, Tumor↗

PubMed2001

Development, standardization, and testing of a lexicon for reporting contrast-enhanced breast magnetic resonance imaging studies.

The purpose of this study was to develop, standardize, and test reproducibility of a lexicon for reporting contrast-enhanced breast magnetic resonance imaging (MRI) examinations. To standardize breast MRI lesion description and reporting, seven radiologists with extensive breast MRI experience developed consensus on technical detail, clinical history, and terminology reporting to describe kinetic and architectural features of lesions detected on contrast-enhanced breast MR images. This lexicon adapted American College of Radiology Breast Imaging and Data Reporting System terminology for breast MRI reporting, including recommendations for reporting clinical history, technical parameters for breast MRI, descriptions for general breast composition, morphologic and kinetic characteristics of mass lesions or regions of abnormal enhancement, and overall impression and management recommendations. To test morphology reproducibility, seven radiologists assessed morphology characteristics of 85 contrast-enhanced breast MRI studies. Data from each independent reader were used to compute weighted and unweighted kappa (kappa) statistics for interobserver agreement among readers. The MR lexicon differentiates two lesion types, mass and non-mass-like enhancement based on morphology and geographical distribution, with descriptors of shape, margin, and internal enhancement. Lexicon testing showed substantial agreement for breast density (kappa = 0.63) and moderate agreement for lesion type (kappa = 0.57), mass margins (kappa = 0.55), and mass shape (kappa = 0.42). Agreement was fair for internal enhancement characteristics. Unweighted kappa statistics showed highest agreement for the terms dense in the breast composition category, mass in lesion type, spiculated and smooth in mass margins, irregular in mass shape, and both dark septations and rim enhancement for internal enhancement characteristics within a mass. The newly developed breast MR lexicon demonstrated moderate interobserver agreement. While breast density and lesion type appear reproducible, other terms require further refinement and testing to lead to a uniform standard language and reporting system for breast MRI. J. Magn. Reson. Imaging 2001;13:889-895.

Breast↗

PubMed2001

Dynamic contrast-enhanced MRI of experimental spinal cord injury: in vivo serial studies.

The progression of experimental spinal cord injury (SCI) was followed with in vivo dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and neurobehavioral studies on postinjury days 0, 2, 4, 7, 10, 14, 17, 21, 28, 35, and 42. Gadopentate dimeglumine (Gd) was administered IV and postcontrast, T(1)-weighted, axial images were acquired repetitively for up to 60 min. Images were analyzed to determine the spatial and temporal evolution of the intensity enhancement. A statistical decision mechanism was developed to objectively detect the enhancement. Strong and rapid enhancement was observed at the epicenter of injury, indicating a significant compromise in blood spinal cord barrier. The enhanced regions in each slice were combined to estimate the area and volume of the lesion. On the day of injury, around 85% of the total cord area at the epicenter showed enhancement within the first 15 min of Gd administration. At the same time, the enhanced volumes attained nearly 40% of the total cord volume and extended axially over 8 mm along the cord. These quantities decreased steadily with time, with a concomitant improvement in the motor functions. The volume of enhancement correlated highly with the neurobehavioral tests (r = -0.87). DCE-MRIs revealed small hyperintense regions distributed inside white matter about two weeks postinjury. Based on histology, these enhancements appear to represent new vessels with "leaky endothelium." Magn Reson Med 45:614-622, 2001.

Animals↗

PubMed2002

Dominant-negative HMGA1 blocks mu enhancer activation through a novel mechanism.

The immunoglobulin mu intronic enhancer is a potent B cell-specific transcriptional activator. The enhancer is activated by the appropriate combination of transcription factors, amongst which are ets and bHLH proteins. HMGA1 (formerly HMG-I(Y)) is a demonstrated co-activator of the mu enhancer. HMGA1 functions through direct interaction with PU.1, one of the ets proteins critical for enhancer activation. New data demonstrates dominant negative HMGA1 dramatically decreases enhancer activity in B cells. EMSA analysis demonstrated that DN HMGA1 disrupts established PU.1/mu enhancer binding. Similarly, DN HMGA1 blocks mu enhancer binding by Ets-1. In sharp contrast, DN HMGA1 had no effect on binding activity of the ETS DNA binding domains of either PU.1 or Ets-1, or the bHLH-zip protein TFE3, suggesting specificity. Taken together, the data suggest that DN HMGA1 utilizes a novel mechanism to specifically block interaction between ets proteins and mu enhancer DNA, suggesting DN HMGA1 represents a new, highly specific means of regulating mu enhancer activity.

Animals↗

PubMed2000

An Nkx-dependent enhancer regulates cGATA-6 gene expression during early stages of heart development.

The evolutionarily conserved GATA-6 transcription factor is an early and persistent marker of heart development in diverse vertebrate species. We previously found evidence for a functionally conserved heart-specific enhancer upstream of the chicken GATA-6 (cGATA-6) gene and in the present study we used transgenic mouse assays to further characterize this regulatory module. We show that this enhancer is activated in committed precursor cells within the cardiac crescent and that it remains active in essentially all cardiogenic cells through the linear heart stage. Although this enhancer can account for cGATA-6 gene expression early in the cardiogenic program, it is not able to maintain expression throughout the heart later in development. In particular, the enhancer is sequentially downregulated along the posterior to anterior axis, with activity becoming confined to outflow tract myocardium. Enhancers with similar properties have been shown to regulate the early heart-restricted expression of the mouse Nkx2.5 transcription factor gene. Whereas these Nkx2.5 enhancers are GATA-dependent, we show that the cGATA-6 enhancer is Nkx-dependent. We speculate that these enhancers are silenced to allow GATA-6 and Nkx2.5 gene expression to be governed by region-specific enhancers in the multichambered heart.

Animals↗

PubMed2002

False-positive helical CT findings of multifocal and multicentric breast cancer: is attenuation of tumor useful for diagnosing enhanced lesions?

OBJECTIVE: To analyze helical CT false-positive multifocal breast cancers and to assess the relevance of the attenuation of tumors for diagnosing enhanced lesions. METHODS: Helical CT studies of 156 invasive breast cancers before breast conserving surgery were examined. A lesion was defined as positive if focal enhancement was detected by CT within 100 seconds after contrast material administration. The attenuation and enhancement percent ratio [(post-contrast value/pre-contrast value)%] were obtained. Attenuation of false-positive and malignant lesions was compared. RESULTS: Helical CT enabled the detection of all 156 invasive tumors with 95 intraductal tumor extensions. The sensitivity and specificity of multifocal/multicentric disease detection by helical CT were 69% and 90%, respectively. False-positive multifocal/multicentric findings were obtained in 11 (7%) of 156 cases. The mean value of the enhancement percent ratio of the index tumors was 237%. Significant differences in the attenuation on post-contrast enhanced scans between the enhanced lesions (index tumors; mean, 82 HU), the true-positive multifocal/multicentric lesions (mean, 73 HU), the false-positive multifocal/multicentric lesions (mean, 87 HU) and normal breast tissue (mean, 32 HU) were found (p<0.0001). The attenuation of the true-positive multifocal/multicentric lesions on post-contrast enhanced scans was significantly less than that of the false-positive multifocal/multicentric lesions (p=0.03). CONCLUSION: Attenuation of tumor is not useful for differential diagnosis of enhanced lesions on helical CT of the breast. The presence of enhancement alone does not always indicate a malignant lesion.

Adult↗

PubMed2004

Dural enhancement with primary calvarial lesions.

The purpose of this study was to relate the pathological and imaging features of dural enhancement and meningeal sign ("dural tail") on contrast-enhanced T1-weighted magnetic resonance (MR) images from patients with primary calvarial lesions as well to assess the accuracy of MR imaging in predicting dural invasion. Thirty-two calvarial tumors studied with contrast-enhanced MR imaging and histopathological examination of the dural specimens were reviewed. Sixteen patients presented dural enhancement, eight with tumor invasion. Tumoral invasion of the dura was observed in one case without enhancement. Malignant lesions showed enhanced dura more commonly than benign lesions (P=0.02). Nodular and discontinuous dural enhancement was statistically associated with dural invasion (P=0.05). Dural tail did not show a specific pathological association. Meningeal enhancement is a nonspecific reaction to calvarial lesions unless nodular and discontinuous. False-negative and -positive cases of dural invasion imply some limitation of contrast-enhanced MR imaging in predicting dural invasion by calvarial neoplasms.

Adolescent↗

PubMed2006

Conservation and divergence of the Emicro3' enhancer in the IGH locus of teleosts.

The core region of the Emicro3' transcriptional enhancer that drives the expression of the teleost IGH locus has been characterized functionally in two species, the catfish (Ictalurus punctatus) and the zebrafish (Danio rerio). These studies have suggested important differences: whereas the catfish enhancer acts through an E-box and two octamer motifs, the zebrafish enhancer exerts its major effects through two E-box motifs alone. In this study, the function of the catfish enhancer was reexamined in a broader comparative context within the teleosts. Electrophoretic mobility shift assays of motifs from catfish, zebrafish, and Fugu were conducted to determine their ability to bind catfish E-protein and Oct transcription factors. Transient expression assays were conducted using a region of the catfish core enhancer that includes a newly described hybrid octamer/E-box motif. Sequences homologous to the Emicro3' enhancer region from six teleosts were aligned to determine conserved regions ("phylogenetic footprinting"). These studies allowed the following conclusions to be drawn: (1) The important 3'E-box motif described in the zebrafish corresponds in the homologous region of the catfish enhancer to an Oct motif with a newly described negative regulatory function and (2) Comparison of the Emicro3' enhancer sequences of six teleosts indicates that while a variety of octamer and E-box motifs are found in this region, strict evolutionary conservation of the important functional elements of the teleost Emicro3' enhancer has not occurred.

Animals↗

PubMed2006

Enhanced MR imaging of tenosynovitis of hand and wrist in inflammatory arthritis.

OBJECTIVE: The purpose of this study is to describe the appearance of tenosynovitis in various tendon groups in the wrist and hand and to compare MR enhanced and non-enhanced imaging evaluation of tenosynovitis of hand and wrist in inflammatory arthritis. DESIGN AND PATIENTS: We reviewed 72 MRI studies of hands and wrists, including coronal, axial and sagittal images in 30 consecutive patients with inflammatory arthritis and tenosynovitis. We compared the degree of synovitis on T2-weighted vs contrast-enhanced T1-weighted images, using a predetermined scale. We also measured the extent of tenosynovitis in three dimensions. The tendons were assigned to volar, dorsal, ulnar and radial groups in the wrist and to extensor, flexor and thumb groups in the hand. Degree of tenosynovitis (graded 0-3), cross-sectional area and volume of the inflamed synovium in various tendon groups were then compared by statistical analysis. RESULTS: Review of the medical records revealed the following diagnoses in our patient population: rheumatoid arthritis (n=16), unspecified inflammatory polyarthritis (n=9), psoriatic arthritis (n=2), CREST syndrome (n=1), systemic lupus erythematosus (n=1), paraneoplastic syndrome with arthritis (n=1). The average T2 brightness scores and post-gadolinium enhancement scores were 1.0 and 1.7, respectively (P<0.001) in the wrist studies. The average T2 brightness scores and post-gadolinium enhancement scores were 0.7 and 1.4, respectively (P<0.001) in the hand studies. The average sensitivity of T2-weighted imaging for detection of tenosynovitis was 40% in the hand and 67% in the wrist tendons, when contrast-enhanced images were used as a reference. Carpal tunnel flexor tendons were the most frequently affected tendons of the wrist. The most frequently affected tendons of the hand were second and third flexor tendons. The hand flexors demonstrated higher degrees of enhancement and larger volumes of the inflamed tenosynovium than did the hand extensors and tendons of the thumb. CONCLUSION: Enhanced MR imaging of the hand and wrist is a superior technique for detection of tenosynovitis. We observed carpal tunnel flexor tendons to be the most frequently affected tendons of the wrist. The flexor tendons of the second and third digits were the most frequently affected tendons of the hands. Higher contrast-enhancement scores and inflammation were noted in the hand flexor than in the extensor tendons.

Adult↗

PubMed2006

[Enhancement between self-realization and self-deception].

DEFINITION OF THE PROBLEM: Is it morally responsible to enhance human beings technically? To answer this question three problems will be analyzed: 1) Do we destroy human nature if we allow enhancement? 2) Can enhancement lead to a loss of autonomy and personality? 3) What are the differences between enhancement of adults and enhancement of children? Arguments and conclusion: I will defend a certain kind of moderate liberalism in order to solve these problems. I will focus on the question whether reversible enhancements must be evaluated differently from irreversible enhancements. Arguments from J. Habermas against enhancement of children will be rejected. Under certain conditions enhancement is not morally unacceptable but it must be ensured that these conditions are fulfilled.

Adult↗

PubMed1992

Stimulation of SV40 DNA replication by the human c-myc enhancer.

In earlier studies we had shown that a transcriptional enhancer sequence exists about 2 kb upstream of the human c-myc gene. The core sequence necessary for enhancer activity was defined therein as a 21 bp nucleotide element, which also showed autonomous replicating activity [EMBO J. (1988) 7, 3135-3142; EMBO J. (1989) 8, 4273-4279]. Recently, several reports have substantiated the notion that transcription and replication can be concertedly regulated in a larger number of cases than expected. In this report, we took the simian virus 40 (SV 40) ori/promoter as a model system. The SV40 enhancer is known to enhance transcription from its ori/promoter, but to reduce its replication (probably due to a negative feedback). The SV40 enhancer was replaced by the c-myc enhancer core in order to see its effect upon SV40 DNA replication and transcription. The results showed that besides stimulating transcription, the c-myc enhancer promoted SV40 DNA replication in monkey CosI cells. Stimulation was only observed when the c-myc enhancer was inserted in the 'up-to-down' orientation to the SV40 promoter. The promoting function of the c-myc enhancer on DNA replication correlated with the transcriptional activation function, as determined by systematic point mutations introduced within the 21 bp core sequence.

Animals↗

PubMed1986

The noncoding region of HPV-6vc contains two distinct transcriptional enhancing elements.

HPV-6vc subgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT) and assayed for the presence of transcriptional enhancing elements. A transcriptional enhancing element was detected in the noncoding region (NCR) of the HPV-6vc viral genome when the CAT assays were performed in viral transformed human kidney cell lines (293 and 324K), in human cervical carcinoma cell lines (HeLa and Siha), and in bovine papillomavirus type 1 (BPV-1) transformed mouse cells (C127-53). The NCR region of the HPV-6b genome was only capable of enhancing transcription of the CAT gene in the HeLa cell line at a level one-third that of the HPV-6vc NCR. The HPV-6vc NCR enhancing activity in C127-53 cells was further stimulated by the addition of sodium butyrate to the growth medium. Localization of the DNA sequences in the HPV-6vc NCR responsible for enhancing transcription revealed two distinct enhancer elements. One element (HPV-6vc position 7218-7544) was active in the 293, HeLa, Siha, and C127-53 cells. The second enhancer element (HPV-6vc position 7544-7971) was only capable of stimulating transcription in HeLa, C127-53, and Siha cells. When the HPV NCR-CAT expression vectors were cotransfected with a competitor plasmid (pNCR75) into C127-53 or HeLa cells then transcriptional enhancement decreased, indicating competition of cellular factors which affect both segments of the HPV-6vc NCR.

Acetyltransferases↗

PubMed1989

Inefficient expression in rat cells of transforming gene of BK virus with 68-bp tandem repeats in the viral promoter-enhancer.

Large-plaque BK virus (wt-501), which has three 68-bp tandem repeats in the viral promoter-enhancer, is not transforming for rat cells, whereas the minute-plaque mutant (dl-504) with only one 68-bp element is. We tested the BKV wt-501 and dl-504 promoter-enhancers for their ability to activate the heterologous and homologous genes in rat 3Y1 cells. The 501 promoter-enhancer activated expression of the chloramphenicol acetyltransferase gene and the neomycin-resistance gene more efficiently than the 504 promoter-enhancer. The 501 enhancer could not allow the efficient expression of BK virus T-antigen gene, whereas the 504 enhancer could. The wt-BKV enhancer with three 68-bp repeats seemed to function positively on the heterologous genes but negatively on the homologous transforming gene in rat cells. The sequence causing the suppressive effects by duplication was confined within the 29-bp segment containing two SV40 enhancer core homologies in the 68-bp element. The results show that inability of wt-BKV to transform rat cells is ascribable to inefficient T-antigen gene expression caused by the duplication in the enhancer and by the surrounding or downstream DNA sequence.

Animals↗

PubMedDate not supplied

The human lambda immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements.

We have recently reported the localization of the first transcriptional enhancer in the human lambda (lambda) immunoglobulin light chain locus. Enhancer activity was contained on a 1.2 kb SstI fragment, with partial activity retained on a core 111 bp PstI-SstI fragment. This enhancer is located 11.7 kb downstream of C lambda 7, the most 3' lambda constant region gene. Using a chloramphenicol acetyl transferase (CAT) assay system, we have now determined the boundaries of the complete enhancer and find it is two- to four-fold as active as the core fragment in both pre-B and B cell lines. Interestingly, a larger fragment, containing the complete enhancer as well as 5' and 3' flanking sequences has four- to eight-fold reduced activity when tested in pre-B cell lines, but full activity in B cell lines. This suggests the presence of developmentally regulated negative elements flanking the human lambda enhancer which prevent or reduce its activity at a developmentally incorrect time. By using in vivo footprinting we have begun to examine the protein interactions within this enhancer in a more physiologically relevant manner and have identified motifs which are shared with the murine lambda enhancers, as well as motifs unique to the human lambda enhancer.

Animals↗