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Results for “Enhancer Elements, Genetic”

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PubMed2001

Modulation of human cytomegalovirus immediate-early gene enhancer by mitogen-activated protein kinase kinase kinase-1.

The immediate-early (IE) promoter of human cytomegalovirus (HCMV) constitutes a primary genetic switch, which determines the progression of viral infection. Earlier reports by others have shown mitogen-activated protein kinase kinase kinase-1 (MEKK1) to be able to up-regulate HCMV-IE promoter through downstream mitogen-activated protein kinase (MAPK) pathways. However, we noticed that the activation of the HCMV-IE promoter by constitutively active MEKK1 (MEKK1-TRU) might not be through the MAPK pathways. Using a HCMV-IE enhancer/promoter (- 522 to + 72) driving a luciferase reporter, we demonstrated that the downstream MAPK activation actually repressed the up-regulation of the promoter by MEKK1 in CHO-K1 and human 293 cells. We further found that the up-regulation of HCMV-IE promoter by MEKK1 could be in great extent suppressed by over-expression of IkappaBalpha. Deletion of the NFkappaB/rel sites in the HCMV-IE enhancer region by mutagenesis proportionally reduced the transcriptional activation by MEKK1-TRU, whereas deletion of the ATF/CREB binding sites or cyclic AMP response elements (CRE) had no effects. Furthermore, the NFkappaB/rel deletion mutant also showed repression on the basic transcription activity of the HCMV-IE promoter. Our results indicate that the NFkappaB/rel sites are not only responsible for the modulation of HCMV-IE enhancer activity by MEKK1 but also control the basic transcription activity of the HCMV-IE promoter. On the other hand, the four consensus CRE sites were found to have no function in the activation of the promoter by MEKK1.

Animals↗

PubMed2004

Identification of a novel polymorphic enhancer of the human CYP3A4 gene.

CYP3A4, the most abundant form of cytochrome P450 in the human adult liver, shows wide interindividual variation in its activity. This variability is thought to be caused largely by transcriptional and genetic factors, yet the underlying mechanisms are poorly understood. The purpose of this study was to clarify the mechanisms controlling the CYP3A4 gene transcription and to search for genetic polymorphisms in the 5'-flanking region of the CYP3A4 gene. Transient transfection of human hepatoma HepG2 cells and of normal human hepatocytes with a series of CYP3A4 promoter-luciferase reporter plasmids revealed that a region from -11.4 to -10.5 kilobases, designated the constitutive liver enhancer module of CYP3A4 (CLEM4), was important for the constitutive activation of the CYP3A4 gene. Gel shift assay using nuclear extracts prepared from HepG2 cells showed that HNF-1alpha, HNF-4alpha, USF1, and AP-1 interacted with CLEM4. Furthermore, the introduction of mutations into their binding sites demonstrated that essentially all sites were required for the maximal enhancer activity. Screening for genetic polymorphisms within CLEM4 in genomic DNA from French persons, we identified the novel variant, TGT insertion between -11,129 and -11,128 (-11,129_-11,128insTGT), whose allele frequency was 3.1%. The -11,129_-11,128insTGT resulted in the disruption of USF1 binding and a 36% reduction of the enhancer activity. These results suggest that CLEM4 is a constitutive enhancer of the CYP3A4 gene in the liver and that -11,129_-11,128insTGT may at least partly contribute to the interindividual variability of CYP3A4 expression.

5' Flanking Region↗

PubMed2003

Human beta-globin locus control region HS5 contains CTCF- and developmental stage-dependent enhancer-blocking activity in erythroid cells.

The human beta-globin locus contains five developmentally regulated beta-type globin genes. All five genes depend on the locus control region (LCR), located at the 5' end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human beta-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the beta-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the epsilon-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the beta-globin gene.

Animals↗

PubMed1999

The FeLV-945 LTR confers a replicative advantage dependent on the presence of a tandem triplication.

Feline leukemia virus (FeLV), like other naturally occurring retroviruses, is characterized by a high degree of genetic diversity. FeLV-945 is a natural isolate derived from non-B-cell non-T-cell lymphomas classified anatomically as multicentric. FeLV-945 exhibits a unique structural motif in the LTR composed of a 21-bp tandem triplication downstream of a single copy of enhancer. The unique FeLV-945 LTR is precisely conserved among eight independent multicentric lymphomas collected in a geographic cluster. Previous studies using reporter gene constructs predict that the FeLV-945 LTR would confer a replicative advantage on the virus that contains it, particularly in primitive hematopoietic cells. Such an advantage may account for the precise conservation of the unique LTR sequence. To test that prediction, a set of recombinant, infectious FeLVs was developed that are isogenic other than the presence of the FeLV-945 LTR or mutations of it. Replication assays show that the FeLV-945 LTR confers a distinct growth advantage in K-562, FEA, and 3201 cells and implicate the 21-bp triplication in that function. Replacement of two copies of the triplicated element with random sequence greatly diminished the replicative capacity, thus implicating the triplicated sequence itself in LTR function. The 21-bp triplication was shown to contain specific nuclear protein binding sites, which may account for the selective pressure to conserve the sequence.

Animals↗

PubMed1990

Genetic suppression of mutations in the Drosophila abl proto-oncogene homolog.

The Drosophila abelson (abl) gene encodes the homolog of the mammalian c-abl cytoplasmic tyrosine kinase and is an essential gene for the development of viable adult flies. Three second-site mutations that suppress the lethality caused by the absence of abl function have been isolated, and all three map to the gene enabled (ena). The mutations are recessive embryonic lethal mutations but act as dominant mutations to compensate for the neural defects of abl mutants. Thus, mutations in a specific gene can compensate for the absence of a tyrosine kinase.

Animals↗

PubMed2004

An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene.

The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.

Alternative Splicing↗

PubMed2003

Brn3a regulation of TrkA/NGF receptor expression in developing sensory neurons.

The TrkA/NGF receptor is essential for the survival and differentiation of sensory neurons. The molecular mechanisms regulating tissue and stage-specific expression of TrkA are largely unknown. The Brn3a POU-domain transcription factor has been implicated in the development of the PNS and proposed as a transcription regulator for TrkA. The molecular mechanisms underlying the regulation of TrkA by Brn3a is unclear. In this study, we provide genetic, transgenic and biochemical evidence that Brn3a binds to novel, specific sites in the 457 bp enhancer that regulates TrkA expression in embryonic sensory neurons. We employ Bax-knockout mice, in which sensory neurons no longer require neurotrophins for survival, to uncouple TrkA-dependent cell death from downregulation of TrkA expression. In addition, when mutagenized, the novel Brn3a-binding sites identified fail to drive appropriate reporter transgene expression in sensory neurons. Thus, TrkA, a gene that is crucial for the differentiation and survival of sensory nociceptive neurons, requires Brn3a to maintain normal transcriptional activity.

Binding Sites↗

PubMed2002

Meis homeoproteins directly regulate Pax6 during vertebrate lens morphogenesis.

Pax6 is a pivotal regulator of eye development throughout Metazoa, but the direct upstream regulators of vertebrate Pax6 expression are unknown. In vertebrates, Pax6 is required for formation of the lens placode, an ectodermal thickening that precedes lens development. Here we show that the Meis1 and Meis2 homeoproteins are direct regulators of Pax6 expression in prospective lens ectoderm. In mice, Meis1 and Meis2 are developmentally expressed in a pattern remarkably similar to Pax6 and their expression is Pax6-independent. Biochemical and transgenic experiments reveal that Meis1 and Meis2 bind a specific sequence in the Pax6 lens placode enhancer that is required for its activity. Furthermore, Pax6 and Meis2 exhibit a strong genetic interaction in lens development, and Pax6 expression is elevated in lenses of Meis2-overexpressing transgenic mice. When expressed in embryonic lens ectoderm, dominant-negative forms of Meis down-regulate endogenous Pax6. These results contrast with those in Drosophila, where the single Meis homolog, Homothorax, has been shown to negatively regulate eye formation. Therefore, despite the striking evolutionary conservation of Pax6 function, Pax6 expression in the vertebrate lens is uniquely regulated.

Animals↗

PubMed1999

Hypomethylation is necessary but not sufficient for V(D)J recombination within a transgenic substrate.

Although an inverse correlation between CpG methylation and V(D)J recombination has been demonstrated for both artificial substrates and endogenous genes, it is not known whether all hypomethylated targets are competent to rearrange or if other factors are required. We have created several artificial V(D)J recombination substrate transgenes whose methylation can be controlled by breeding into different genetic backgrounds. A transgene which contains the immunoglobulin heavy chain intronic enhancer rearranges efficiently in B lymphocytes when the transgene loci are unmethylated. When the same loci become methylated, upon breeding into a different mouse strain, no rearrangement can be detected. A similar transgene, but lacking the enhancer, also shows no evidence of V(D)J recombination when it is methylated. Even when this enhancerless transgene is hypomethylated, however, no V(D)J recombination can be detected in B lymphocytes. Thus, hypomethylation is required to permit V(D)J recombination but not all hypomethylated targets are capable of recombination. The results may indicate that the immunoglobulin enhancer is required for the assembly of factors involved in V(D)J recombination.

Animals↗

PubMed1997

Genetic relationships between the mutations spade and Sternopleural and the wingless gene in Drosophila development.

In Drosophila melanogaster, there are cases in which gene products contributing to the same developmental event may derive from closely adjacent transcription units and may even share cis-regulatory sequences. Correct recognition of such genomic organization is central to an understanding of developmental mechanisms. The adult phenotypes of combinations between the mutations spade, Sternopleural, and wingless suggest that they are lesions in functionally related genes within the same chromosomal region. wingless mutations fail to complement the recessive mutation spade. The spade mutation, as previously shown, behaves as a lesion in a regulatory site of wingless, sited 5' to the transcription unit, and is concerned with particular postembryonic functions of wingless. While showing wingless-like phenotypes in combination with Sternopleural, even lethal alleles of wingless complement the recessive lethality of Sternopleural alleles. Mutations in Sternopleural increase the severity of wingless phenotypes in many wingless-dependent processes during postembryonic development, and this interaction can occur when the only functional copies of Sp or wg are located in either opposing chromosomes or the same chromosome. This is inconsistent with previous attempts to define Sp as a regulatory allele of wg and explain the phenotypes that result from combinations of Sp and wg by means of transvection. We have analyzed a new EMS-induced allele of Sternopleural that is more severe than the original allele, which also argues for Sp being a separate, mutable genetic locus rather than a regulatory allele of wg. Finally, we have a revertant of Sternopleural (Sp[Rv1]) that behaves as a genetic null allele of wg, but causes ventral-to-dorsal transformations in combination with wg(P), which is not observed in combinations of wg null alleles with wg(P). Because wg(P) is the result of an inversion and because inversions inhibit transvection, the increased severity observed in Sp(Rv1)/wg(P) in comparison to wg(null)/Sp(Rv1) animals cannot be explained by an absence of transvection. Therefore, the two Sternopleural mutations most reasonably define an independent gene located 3' to the wingless gene and having strong functional synergism with it.

Animals↗

PubMed1999

INK4a/ARF mutations accelerate lymphomagenesis and promote chemoresistance by disabling p53.

The INK4a/ARF locus encodes upstream regulators of the retinoblastoma and p53 tumor suppressor gene products. To compare the impact of these loci on tumor development and treatment response, the Emu-myc transgenic lymphoma model was used to generate genetically defined tumors with mutations in the INK4a/ARF, Rb, or p53 genes. Like p53 null lymphomas, INK4a/ARF null lymphomas formed rapidly, were highly invasive, displayed apoptotic defects, and were markedly resistant to chemotherapy in vitro and in vivo. Furthermore, INK4a/ARF(-/-) lymphomas displayed reduced p53 activity despite the presence of wild-type p53 genes. Consequently, INK4a/ARF and p53 mutations lead to aggressive tumors by disrupting overlapping tumor suppressor functions. These data have important implications for understanding the clinical behavior of human tumors.

Animals↗

PubMed1993

A 168 bp derivative of Suppressor-mutator/Enhancer is responsible for the maize o2-23 mutation.

From a directed transposon tagging of the maize Opaque-2 gene (O2), we have isolated a stable mutant o2 allele, o2-23. Cloning and molecular analysis of the allele revealed a 168 nucleotide insertion in the third exon of o2. The sequence of this small insertion indicated identity with the 5' and 3' ends of the 8.3 kb Suppressor-mutator/Enhancer (Spm/En) transposable element. This represents the smallest deletion derivative of Spm (dSpm) thus far characterized in maize. Genetic crosses of plants homozygous for o2-23 with plants homozygous for both an o2 null allele (o2-R) and an autonomous Spm produce stable opaque seed having no apparent sectors of vitreous endosperm. DNA fragments of the size expected if the dSpm were to excise were not detectable by Southern analysis, suggesting that this element is unable to transpose. Northern analysis detected an o2-23 mRNA that was much more abundant in o2-23 seeds lacking Spm than in o2-23 seeds containing Spm, consistent with the idea that Spm transacting functions can suppress the accumulation of the o2-23 transcript.

Alleles↗

PubMed1995

LEUNIG regulates AGAMOUS expression in Arabidopsis flowers.

LEUNIG was identified in a genetic screen designed to isolate second-site enhancer mutations of the floral homeotic mutant apetala2-1. leunig mutations not only enhance apetala2, but by themselves cause a similar but less-pronounced homeotic transformation than apetala2 mutations. leunig flowers have sepals that are transformed toward stamens and carpels, and petals that are either staminoid or absent. In situ hybridization experiments with leunig mutants revealed altered expression pattern of the floral homeotic genes APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Double mutants of leunig and agamous exhibited a phenotype similar to agamous single mutants, indicating that agamous is epistatic to leunig. Our analysis suggests that a key role of LEUNIG is to negatively regulate AGAMOUS expression in the first two whorls of the Arabidopsis flower.

Arabidopsis↗

PubMed2001

Genetic and molecular characterization of a variegating hsp70-acZ fusion gene in the euchromatic 31 B region of Drosophila melanogaster.

A hsp70-lacZ fusion gene introduced into Drosophila melanogaster at the euchromatic 31B region by Pelement transformation displayed a variegated expression with respect to the lacZ fusion protein in the salivary gland cells under heat-shock conditions. The variegation is also reflected by the chromosome puffing pattern. Subsequent transposition of the 31B P element to other euchromatic positions restored wild-type activity, that is, a nonvariegated phenotype. A lower developmental temperature reduced the amount of expression under heat-shock conditions, similar to genes undergoing position-effect variegation (PEV). However, other modifiers of PEV did not affect the expression pattern of the gene. These results show a novel euchromatic tissue-specific variegation that is not associated with classical heterochromatic PEV.

Animals↗

PubMed2006

Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression.

In eukaryotes, the ability of DNA-binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified, including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Here we demonstrate strong genetic interactions between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. We show that mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests that they do not function together, but instead that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity.

Animals↗

PubMed1995

Light and developmental regulation of the gene con-10 of Neurospora crassa.

The gene con-10 of Neurospora crassa is expressed preferentially during conidiation and following illumination of vegetative mycelia with blue light. In this study we have examined the segmental locations of the genetic elements associated with con-10 that are responsible for light and developmental expression. A translational fusion was prepared between the initial segment of con-10 and Escherichia coli lacZ. Deletions were then introduced into the con-10 upstream region associated with this translational fusion. Each construct was integrated at the his-3 locus of N. crassa by transformation and homologous recombination. Photoinduction of mycelia containing the translational fusion with the intact upstream region revealed a two phase stimulus-response curve. Exposure to light for as little as 5 sec induced a transcriptional response. Following this initial induction, a period of 15 min in the dark or light was required for appearance of a second phase response. Only a brief light treatment was necessary for induction of the second phase response. Deletions within the upstream region altered normal light and developmental expression of constructs containing the con-10-lacZ translational fusion. The deleted segments appear to contain a mycelial repression site, two conidiation activation sites, and two dark repression sites. A repeated 17-bp sequence acted as a transcriptional enhancer. One copy of this enhancer is in the upstream region. The second copy, with the opposite orientation, is located in the first con-10 intron. The enhancer was required for proper mycelial and conidial expression of the con-10-lacZ fusion. The initial 10 bp of this enhancer sequence were sufficient to restore conidial expression to a deletion construct lacking both copies of the 17-bp repeat. Proteins were detected in extracts of mycelia and conidia that specifically bound to the enhancer sequence in vitro. Our findings suggest that conidiation-specific and mycelial-specific expression of con-10 requires the action of several factors acting independently and/or in concert at distinct sites located in the regulatory regions for con-10.

Base Sequence↗

PubMed1989

The genetics of position-effect variegation modifying loci in Drosophila melanogaster.

The dose dependent effects of position-effect variegation (PEV) modifying genes were studied in chromosome arms 2L, 2R and 3R. Four groups of PEV modifying genes can be distinguished: haplo-abnormal suppressor and enhancer loci with or without a triplo-effect. Using duplications four triplo-abnormal suppressor and four triplo-abnormal enhancer functions were localized. In two cases we proved that these functions correspond to a converse haplo-abnormal one. Altogether 43 modifier loci were identified. Most of these loci proved not to display significant triplo-effects (35). The group of haplo-abnormal loci with a triplo-effect may represent genes which play an important role in heterochromatin packaging.

Animals↗

PubMed2004

A classical arabinogalactan protein is essential for the initiation of female gametogenesis in Arabidopsis.

Classical arabinogalactan proteins (AGPs) are an abundant class of cell surface proteoglycans widely distributed in flowering plants. We have used a combination of enhancer detection tagging and RNA interference (RNAi)-induced posttrancriptional silencing to demonstrate that AGP18, a gene encoding a classical arabinogalactan protein, is essential for female gametogenesis in Arabidopsis thaliana. AGP18 is expressed in cells that spatially and temporally define the sporophytic to gametophytic transition and during early stages of seed development. More than 75% of the T1 transformants resulted in T2 lines showing reduced seed set during at least three consecutive generations but no additional developmental defects. AGP18-silenced T2 lines showed reduced AGP18 transcript levels in female reproductive organs, the presence of 21-bp RNA fragments specific to the AGP18 gene, and the absence of in situ AGP18 mRNA localization in developing ovules. Reciprocal crosses to wild-type plants indicate that the defect is female specific. The genetic and molecular analysis of AGP18-silenced plants containing a single T-DNA RNAi insertion suggests that posttranscriptional silencing of AGP18 is acting both at the sporophytic and gametophytic levels. A cytological analysis of all defective AGP18-RNAi lines, combined with the analysis of molecular markers acting at key stages of female gametogenesis, showed that the functional megaspore fails to enlarge and mitotically divide, indicating that AGP18 is essential to initiate female gametogenesis in Arabidopsis. Our results assign a specific function in plant development to a gene encoding a classical AGP.

Arabidopsis↗