Mechanism of chromatin recognition and transcriptional regulation by LEF-1 and the Wnt/Wg-responsive LEF-1:beta-catenin complex.
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Amino Acid Sequence↗
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Results for “Enhancer Elements, Genetic”
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Enhancer of garnet/deltaAP-3 is a cryptic allele of the white gene and identifies the intracellular transport system for the white protein.
The white gene encodes an ABC-type transmembrane transporter that has a role in normal eye pigment deposition. In addition, overexpression in Drosophila leads to homosexual male courtship. Its human homologue has been implicated in cholesterol transport in macrophages and in mood disorders in human males. The garnet gene is a member of a group of other Drosophila eye colour genes that have been shown, or proposed, to function in intracellular protein transport. Recent molecular analysis indicates that it encodes the delta subunit of the AP-3 adaptin complex involved in vesicle transport from the trans-Golgi network to lysosomes and related organelles, such as pigment granules. This identification revealed a novel role for intracellular vesicular transport in Drosophila pigmentation. To further analyze this intracellular transport system, we examined the genetic interactions between garnet and a second site enhancer mutation, enhancer of garnet (e(g)). We show here that e(g) is a cryptic allele of the white gene. The white-garnet interaction is highly sensitive to the levels of both gene products but also shows some allele specificity for the white gene. The additive effect on pigmentation and the predicted protein products of these genes suggest that the garnet/AP-3 transport system ensures the correct intracellular localization of the white gene product. This model is further supported by the observation of homosexual male courtship behavior in garnet mutants, similar to that seen in flies overexpressing, and presumably mis-sorting, the white gene product. The w(e(g)) allele also enhances mutations in the subset of other eye-color genes with phenotypes similar to garnet. This observation supports a role for these genes in intracellular transport and leads to a model whereby incorrect sorting of the white gene product can explain the pigmentation phenotypes of an entire group of eye-color genes.
ATP-Binding Cassette Transporters↗
The Drosophila Pox neuro gene: control of male courtship behavior and fertility as revealed by a complete dissection of all enhancers.
We have dissected the entire cis-regulatory region of the Drosophila Pox neuro gene with regard to its enhancers, and have analyzed their functions by the selective addition to Pox neuro null mutant flies of one or several functions, each regulated by a complete or partial enhancer. We have identified at least 15 enhancers with an astounding complexity in arrangement and substructure that regulate Pox neuro functions required for the development of the peripheral and central nervous system and of most appendages. Many of these functions are essential for normal male courtship behavior and fertility. Two enhancers regulate the development of the penis, claspers and posterior lobes of male genitalia. Three enhancers, two of which overlap, control the development of chemosensory bristles in the labellum, legs and wings, some or all of which are required for the transmission of gustatory signals elicited by female pheromones. An additional enhancer regulates in the developing brain the connectivity of two specific neuronal clusters entrusted with processing olfactory pheromone signals from the antennal nerve. Finally, functions crucial for the ability of the male to copulate depend on an enhancer that activates Pox neuro expression in the embryonic ventral cord. In addition to these male courtship and fertility functions of Pox neuro, we have identified enhancers that regulate: (1) proper segmentation of tarsal segments in the leg disc and in homologous segments of the antennal disc; and (2) proper development of the wing hinge and hence the ability of the fly to fly.
Alleles↗
Characterization of enhancer trap and gene trap harboring Ac/Ds transposon in transgenic rice.
Insertion mutagenesis has become one of the most popular methods for gene functions analysis. Here we report a two-element Ac/Ds transposon system containing enhancer trap and gene trap for gene tagging in rice. The excision of Ds element was examined by PCR amplification. The excision frequency of Ds element varied from 0% to 40% among 20 F(2) populations derived from 11 different Ds parents. Southern blot analysis revealed that more than 70% of excised Ds elements reinserted into rice genome and above 70% of the reinserted Ds elements were located at different positions of the chromosome in rice. The result of histochemical GUS analysis indicated that 28% of enhancer trap and 22% of gene trap tagging plants displayed GUS activity in leaves, roots, flowers or seeds. The GUS positive lines will be useful for identifying gene function in rice.
Base Sequence↗
Multiple elements may be used for regulation of the GAP-43 gene in different cell-types.
Recent evidence suggests that GAP-43 expression is not restricted to the nervous system, but may also occur outside the neural cell lineage. Two distinct patterns of GAP-43 regulation can therefore be distinguished. The first is the regulation of GAP-43 expression in multiple cell-types, and the second is the gene's temporal modulation within one specific cell-type. The latter type is well documented for neurons, where GAP-43 regulation is regulated in a fashion that is dependent on axon integrity. Results from partial analysis of the GAP-43 promoter/enhancer region indicate that at least some of these aspects of GAP-43 gene regulation may be accounted for by distinct cis-acting elements. For example, the expression of a rat GAP-43 promoter fusion gene in epidermal cells of transgenic zebrafish is dependent on an enhancer element, that is clearly distinct from the minimal neural-specific promoter. Characterization of specific GAP-43 regulatory elements responsible for particular aspects of its regulation may provide insight to signal pathways also utilized by other genes during development. Ultimately, a better understanding of the molecular events during development could help to define more precisely the complex sequences necessary for the establishment of an intact organism.
Animals↗
Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs.
To investigate the basis for enhancer-promoter specificity, we compared the ability of enhancers to activate transcription in vivo from core promoters containing either downstream promoter element (DPE) or TATA box motifs. To eliminate position effects, we generated and analyzed pairs of sister Drosophila lines that contain a DPE- or TATA-dependent reporter gene at precisely the same genomic position relative to each enhancer. These studies revealed transcriptional enhancers that are specific for promoters that contain either DPE or TATA box elements. Thus, the core promoter not only mediates the initiation of transcription, but also functions as a regulatory element.
Animals↗
Positive and negative cis-acting elements are required for hematopoietic expression of zebrafish GATA-1.
GATA-1 is a transcription factor required for development of erythroid cells. The expression of GATA-1 is tightly restricted to the hematopoietic lineage. Using transgene constructs containing zebrafish GATA-1 genomic sequences and the green fluorescent protein (GFP) reporter gene, we previously showed that a 5.6-kb enhancer/promoter fragment is sufficient to direct erythroid-specific expression of the GFP. In this study, we used enhancer/promoter fragments containing various deletion and point mutations to further characterize the cis-acting elements controlling tissue-specific GATA-1 expression. We report here the identification of distinct cis-acting elements that cooperate to confer on GATA-1 its hematopoietic expression pattern. A CACCC box, located 142 bp upstream of the translation start codon, is critical for the initiation of GATA-1 expression. A distal double GATA element is required for maintaining and enhancing the hematopoietic expression of GATA-1. The erythroid-specific activity of the GATA-1 promoter is also enhanced by a 49-bp sequence element located 218 bp upstream of the CACCC element and a CCAAT box adjacent to the double GATA motif. Finally, the hematopoietic specificity of the GATA-1 promoter is secured by a negative cis-acting element that inhibits expression in the notochord.
Animals↗
Multipartite genetic control elements: communication by DNA loop.
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DNA↗
Regulation of hepatitis B virus gene expression.
Human hepatitis B virus (HBV) mainly infects hepatocytes. HB viral gene expression has been demonstrated to be liver-specific using DNA transfection methods. This liver-specific gene expression is regulated by promoter/enhancer elements. HBV contains two enhancer elements. Enhancer element I has been studied in detail at the DNA-protein level. This is further substantiated by DNA transfections of liver and non-liver cell lines with expression plasmids containing enhancer elements controlling the transcription of reporter genes. Genetic analysis of the enhancer elements defined the minimal sequences which play a key role in the regulation of enhancer function. One of the factors binding in this region is RXR alpha. Using only the DNA binding domain of the liver-specific RXR alpha expressed in E. coli, we demonstrated binding of RXR alpha to the putative retinoic acid receptor response element (RARE) in the HBV enhancer. Our studies implicate a potentially important role of retinoic acid and its receptor in the liver-specific regulation of HBV gene expression and the disease pathogenesis associated with infection.
Base Sequence↗
Interaction of the Enhancer of white-apricot with transposable element alleles at the white locus in Drosophila melanogaster.
The Enhancer of wa [E(wa)] mutation was shown to interact strongly with 4 of 41 tested alleles of the white (w) eye color locus. All four of the affected w alleles result from the insertion of a transposable element. E(wa) was further localized cytogenetically. The locus lies between the breakpoints of T(Y;2)L11 and T(Y;2)H137 (section 60) in 2R. The original mutation was shown to be antimorphic on the basis of its action in the presence of additional normal copies and the ability to revert the original allele to one that mimics the effect of a deficiency for the locus. The RNA transcribed from wa was analyzed from flies segregating for E(wa) and normal. The low level of normal functional messenger RNA present in white-apricot is reduced further in Enhancer homozygotes. Total copia RNA was also examined on Northern analyses from the segregating population but no quantitative change in the major copia RNA was produced by E(wa) homozygotes compared to normal.
Alleles↗
The LIM domain homeobox gene isl-1 is a positive regulator of islet cell-specific proglucagon gene transcription.
The LIM domain homeobox gene islet 1 (isl-1) is expressed in the embryonic nervous system and may be an early marker of motor neuron specification. isl-1 is expressed in all 4 islet cell types, but a role for isl-1 in the regulation of insulin gene expression has not been demonstrated, and the genetic targets for isl-1 in the pancreas remain unknown. We show here that the proximal rat proglucagon gene promoter binds an amino-terminally truncated Trp-E-isl-1 fusion protein that lacks the LIM domains. The proglucagon gene promoter also binds full-length in vitro translated isl-1 containing the intact LIM domains. isl-1 antisera detects binding of proglucagon gene sequences to isl-1 present in a slowly-migrating complex in nuclear extracts from InR1-G9 islet cells. The transcriptional properties of the proglucagon gene promoter sequences that bind isl-1 (designated Ga, Gb, and Gc) were assessed after transfection of reporter genes into wild type and isl-1-antisense (isl-1(AS)) InR1-G9 islet cells. The proximal proglucagon gene (Ga) promoter sequence reduced TK-CAT activity by approximately 50%, but no change in the activity of the Ga-TK-CAT plasmid was seen after transfection of isl-1(AS) InR1-G9 cells. In contrast, the Gb/Gc sites activated transcription 2-3 fold in wild type InR1-G9 cells, and the isl-1-dependent activation of gene transcription through the Gb/Gc element was eliminated following transfection of isl-1(AS) InR1-G9 cells. These data demonstrate that the LIM domain homeobox gene isl-1 1) is not constrained from DNA binding by its LIM domains and 2) functions as a positive regulator of proglucagon gene transcription in the endocrine pancreas.
Animals↗
Identification of a purine-rich intronic enhancer element in the mouse eosinophil-associated ribonuclease 2 (mEar 2) gene.
The Mus musculus eosinophil-associated ribonuclease (mEar) gene cluster includes multiple distinct coding sequences that are highly divergent orthologs of the human eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3). We present a transcriptional analysis of the gene encoding mEar 2, the only member of this cluster with a well-defined expression profile. In this work, we demonstrate that the presence of non-coding exon 1 and the intron in tandem with a 361-bp 5' promoter of mEar 2 results in enhanced reporter gene expression, as much as 6-to 10-fold over the activity observed with the 5' promoter alone. We have identified a conserved purine-rich element in the intron of the mEar 2 gene that is necessary for maximum transcription and that interacts specifically with NFAT-binding proteins in nuclear extracts derived from the mouse LA4 epithelial cell line. Similar intronic enhancers have been described as regulating transcription of the human EDN gene, suggesting an overall conservation of an important regulatory strategy.
Animals↗
The gypsy insulators flanking yellow enhancers do not form a separate transcriptional domain in Drosophila melanogaster: the enhancers can activate an isolated yellow promoter.
The best-characterized insulator in Drosophila melanogaster is the Su(Hw)-binding region contained within the gypsy retrotransposon. In the y(2) mutant, Su(Hw) protein partially inhibits yellow transcription by blocking the function of transcriptional enhancers located distally from the yellow promoter with respect to gypsy. Previously we have shown that yellow enhancers can overcome inhibition by a downstream insulator in the y(rh1) allele, when a second gypsy element is located upstream of the enhancers. To understand how two insulators neutralize each other, we isolated various deletions that terminate in the regulatory region of the y(rh1) allele. To generate these alleles we used DNA elongation by gene conversion of the truncated chromosomes at the end of the yellow regulatory region. We found that gypsy insulator can function at the end of the truncated chromosome. Addition of the gypsy insulator upstream of the yellow enhancers overcomes the enhancer-blocking activity of the gypsy insulator inserted between the yellow enhancers and promoter. These results suggest that the gypsy insulators do not form separate transcriptional domains that delimit the interactions between enhancers and promoters.
Animals↗
GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo.
Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The eve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl-GAGA increases the stability of enhancer-promoter interactions by creating an open chromatin configuration at the core promoter.
Animals↗
An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues.
The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.
Animals↗
Differences in insulator properties revealed by enhancer blocking assays on episomes.
Insulators are genomic elements that define domains of transcriptional autonomy. Although a large number of insulators have been isolated, it is unclear whether these elements function by shared molecular mechanisms. Novel applications of FLP recombinase technology were used to dissect and compare the function of the Drosophila: gypsy and scs insulators. Inter actions between FLP monomers bound to chromosomally integrated FRT sites were unimpeded by either insulator, demonstrating that these insulators do not establish a chromosomal environment capable of disrupting all types of protein-protein interactions. The gypsy insulator blocked enhancer-activated transcription on FLP-generated extra-chromosomal episomes, whereas the scs insulator displayed silencing effects. These data indicate that these insulators differ in the mechanisms used to prevent enhancer function. That the gypsy insulator blocked enhancer-promoter communication within small episomes suggests that these effects may be accomplished without a global reorganization of chromatin structure. Instead, the gypsy insulator may disrupt enhancer-activated transcription by direct interference with transmission of the enhancer signal to the promoter.
Animals↗
LTR retrotransposons and the evolution of eukaryotic enhancers.
Since LTR retrotransposons and retroviruses are especially prone to regional duplications and recombination events, these viral-like systems may be especially conducive to the evolution of closely spaced combinatorial regulatory motifs. Using the Drosophila copia LTR retrotransposon as a model, we show that a regulatory region contained within the element's untranslated leader region (ULR) consists of multiple copies of an 8 bp motif (TTGTGAAA) with similarity to the core sequence of the SV40 enhancer. Naturally occurring variation in the number of these motifs is correlated with the enhancer strength of the ULR. Our results indicate that inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and perhaps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to play a role in regulatory evolution.
Animals↗
Identification of a novel enhancer element mediating calcium-dependent induction of gene expression in response to either epidermal growth factor or activation of protein kinase C.
The VL30 family of defective murine retroviruses consists of 100 to 200 members, of which fewer than 5% appear to be transcriptionally active. A genomic clone of the transcriptionally active VL30 element RVL-3 was identified and sequenced. Genetic analysis indicated that a triple-repeat sequence within the RVL-3 long terminal repeat is capable of functioning as an inducible enhancer element responding to a variety of agonists. In Rat-1 fibroblasts, the ability of the RVL-3 enhancer to mediate induction of gene expression from a heterologous promoter in response to either epidermal growth factor (EGF) or phorbol ester treatment required coelevation of intracellular calcium. Two CArG boxes present in the triple-repeat sequence appeared to exert a negative effect on gene expression, as mutation of these sequences elevated the basal level of expression observed without altering the fold induction in response to either EGF or protein kinase C activation. In the presence of these CArG elements, mutation of AP-1-like sites adjacent to the CArG elements significantly inhibited the ability of either EGF or phorbol esters to induce gene expression. The effect of mutating these AP-1-like sites was relieved by simultaneous mutation of the CArG sites, indicating that interactions among these sites modulate RVL-3 expression. Mutational analysis and gel mobility shift experiments have identified a third sequence within the VL30 triple-repeat element that is required for the induction of gene expression and serves as a binding site for nuclear proteins. Sequence comparisons indicate that this enhancer element has not been described previously.
Animals↗