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Results for “Enhancer Elements, Genetic”

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PubMed1985

Comparison of the transcriptional properties of the Friend and Moloney retrovirus long terminal repeats: importance of tandem duplications and of the core enhancer sequence.

The effects of sequence differences within the long terminal repeats (LTRs) of various murine retroviruses on transcription are examined by linking genetically engineered recombinant LTRs to the protein coding region of the herpes simplex virus thymidine kinase (TK) gene and assaying TK expression in tissue culture fibroblasts. The Goldberg/Hogness box region and the enhancer region are examined independently. We find that the Friend and Moloney Goldberg/Hogness boxes (TATAAAA and AATAAAA, respectively) give similar results in this assay, whereas differences between the sequences in the enhancer region have a marked effect. Tandem duplications increase the transcription level of the LTR. A single nucleotide transition in the core enhancer sequence has as large an effect on transcription as the presence of a tandem duplication: thymidine in the fifth position of the core enhancer (TCTGTGGTAAG) leads to a much higher transcriptional activity than cytidine. The LTRs have been implicated by others in the oncogenic potential of murine retroviruses, which is perhaps dependent on the transcriptional properties endowed in part by the core enhancer and tandem duplications.

Base Sequence↗

PubMed1998

Identification of an N-terminal region of sigma 54 required for enhancer responsiveness.

Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.

Amino Acid Sequence↗

PubMed2000

Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter.

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542)

Actins↗

PubMed1996

Identification and characterization of autosomal genes that interact with glass in the developing Drosophila eye.

The glass gene encodes a zinc finger, DNA-binding protein that is required for photoreceptor cell development in Drosophila melanogaster. In the developing compound eye, glass function is regulated at two points: (1) the protein is expressed in all cells' nuclei posterior to the morphogenetic furrow and (2) the ability of the Glass protein to regulate downstream genes is largely limited to the developing photoreceptor cells. We conducted a series of genetic screens for autosomal dominant second-site modifiers of the weal allele glass3, to discover genes with products that may regulate glass function at either of these levels. Seventy-six dominant enhancer mutations were recovered (and no dominant suppressors). Most of these dominant mutations are in essential genes and are associated with recessive lethality. We have assigned these mutations to 23 complementation groups that include multiple alleles of Star and hedgehog as well as single alleles of Delta, roughened eye, glass and hairy. Mutations in 18 of the complementation groups are embryonic lethals, and of these, 13 show abnormal adult retinal phenotypes in homozygous clones, usually with altered numbers of photoreceptor cells in some of the ommatidia.

Animals↗

PubMed1999

Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

Animals↗

PubMed1996

Genetic interactions affecting touch sensitivity in Caenorhabditis elegans.

At least 13 genes (mec-1, mec-2, mec-4-10, mec-12, mec-14, mec-15, and mec-18) are needed for the response to gentle touch by 6 touch receptor neurons in the nematode Caenorhabditis elegans. Several, otherwise recessive alleles of some of these genes act as dominant enhancer mutations of temperature-sensitive alleles of mec-4, mec-5, mec-6, mec-12, and mec-15. Screens for additional dominant enhancers of mec-4 and mec-5 yielded mutations in previously known genes. In addition, some mec-7 alleles showed allele-specific, dominant suppression of the mec-15 touch-insensitive (Mec) phenotype. The dominant enhancement and suppression exhibited by these mutations suggest that the products of several touch genes interact. These results are consistent with a model, supported by the known sequences of these genes, that almost all of the touch function genes contribute to the mechanosensory apparatus.

Alleles↗

PubMed2004

Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication.

The 5' portion of the Sindbis virus (SIN) genome RNA is multifunctional. Besides initiating translation of the nonstructural polyprotein, RNA elements in the 5' 200 bases of the SIN genome RNA, or its complement at the 3' end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand RNAs. We used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. Replacement of the SIN 5' end in defective-interfering (DI) and genome RNAs with sequences from a distantly related alphavirus, Semliki Forest virus (SFV), resulted in nonviable chimeras. The addition of five nucleotides from the 5' terminus of SIN restored negative-strand RNA synthesis in DI genomes but not their replication in vivo. Pseudorevertants of various SFV-SIN chimeras were isolated, and suppressor mutations were mapped to AU-rich sequences added to the 5' end of the original SFV 5' sequence or its "deleted" versions. Early pseudorevertants had heterogeneous 5' termini that were inefficient for replication relative to the parental SIN 5' sequence. In contrast, passaging of these pseudorevertant viral populations in BHK cells under competitive conditions yielded evolved, more homogeneous 5'-terminal sequences that were highly efficient for negative-strand synthesis and replication. These 5'-terminal sequences always began with 5'-AU, followed by one or more AU repeats or short stretches of oligo(A). Further analysis demonstrated a positive correlation between the number of repeat units and replication efficiency. Interestingly, some 5' modifications restored high-level viral replication in BHK-21 cells, but these viruses were impaired for replication in the cells of mosquito origin. These studies provide new information on sequence determinants required for SIN RNA replication and suggest new strategies for restricting cell tropism and optimizing the packaging of alphavirus vectors.

5' Untranslated Regions↗

PubMed1996

Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure.

We have analyzed the dioxin-inducible transcriptional control mechanism for the mouse CYP1A1 gene in its native chromosomal context. Our genetic and biochemical studies indicate that a C-terminal segment of the aromatic hydrocarbon receptor (AhR) contains latent transactivation capability and communicates the induction signal from enhancer to promoter. Thus, transactivation and enhancer-promoter communication may be congruent functions of AhR. Both functions require heterodimerization between AhR and the AhR nuclear translocator (Arnt). Our findings also indicate that heterodimerization activates AhR's latent transactivation function and silences that of Arnt. Furthermore, removal of Arnt's transactivation domain does not affect dioxin-induced CYP1A1 transcription in vivo. In addition, our studies demonstrate that dioxin-induced changes in chromatin structure occur by different mechanisms at the CYP1A1 enhancer and promoter and that events at an enhancer can be experimentally dissociated from events at the cognate promoter during mechanistic analyses of mammalian transcription in vivo.

Animals↗

PubMed1989

Influence of enhancer sequences on thymotropism and leukemogenicity of mink cell focus-forming viruses.

Oncogenic mink cell focus-forming (MCF) viruses, such as MCF 247, show a positive correlation between the ability to replicate efficiently in the thymus and a leukemogenic phenotype. Other MCF viruses, such as MCF 30-2, replicate to high titers in thymocytes and do not accelerate the onset of leukemia. We used these two MCF viruses with different biological phenotypes to distinguish the effect of specific viral genes and genetic determinants on thymotropism and leukemogenicity. Our goal was to identify the viral sequences that distinguish thymotropic, nonleukemogenic viruses such as MCF 30-2 from thymotropic, leukemogenic viruses such as MCF 247. We cloned MCF 30-2, compared the genetic hallmarks of MCF 30-2 with those of MCF 247, constructed a series of recombinants, and tested the ability of recombinant viruses to replicate in the thymus and to induce leukemia. The results established that (i) MCF 30-2 and MCF 247 differ in the numbers of copies of the enhancer sequences in the long terminal repeats. (ii) The thymotropic phenotype of both viruses is independent of the number of copies of the enhancer sequences. (iii) The oncogenic phenotype of MCF 247 is correlated with the presence in the virus of duplicated enhancer sequences or with the presence of an enhancer with a specific sequence. These results show that the pathogenic phenotypes of MCF viruses are dissociable from the thymotropic phenotype and depend, at least in part, upon the enhancer sequences. On the basis of these results, we suggest that the molecular mechanisms by which the enhancer sequences determine thymotropism are different from those that determine oncogenicity.

Amino Acid Sequence↗

PubMed2005

Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors.

Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR) is copied over into the 5' LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.

Animals↗

PubMed2025

Systematic decoding of functional enhancer connectomes and risk variants in human glioma.

Genetic and epigenetic variations contribute to the progression of glioma, but the mechanisms underlying these effects, particularly for enhancer-associated genetic variations in non-coding regions, still remain unclear. Here we performed high-throughput CRISPR interference screening to identify pro-tumour enhancers in glioma cells. By integrating genome-wide H3K27ac HiChIP data, we identified the target genes of these pro-tumour enhancers and revealed the essential role of enhancer connectomes in promoting glioma progression. Through systematic analysis of enhancers carrying glioma risk-associated single-nucleotide polymorphisms (SNPs), we found that these SNPs can promote glioma progression through the enhancer connectome. Using CRISPR-Cas9-mediated enhancer interference and SNP editing, we demonstrated that glioma-specific enhancer carrying the risk SNP rs2297440 regulates SOX18 expression by specifically recruiting transcription factor MEIS1 binding, thereby contributing to glioma progression. Our study sheds light on the molecular mechanisms underlying glioma susceptibility and provides potential therapeutic targets to treat glioma.

Humans↗

PubMed1986

Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA.

We isolated and sequenced 6.3 kb of cDNA encoding that rat glucocorticoid receptor, a protein that binds and activates a class of hormone-dependent transcriptional enhancers. Receptor-containing cells produce receptor mRNAs of approximately equal to 6.5 kb and approximately equal to 4.8 kb that differ only in their 3' nontranslated regions; an open reading frame of 795 amino acids resides within the 5' portion of the transcripts. The coding region was expressed in vitro, in transient transfections, and in stable transfectants of a receptor-deficient cell line. The protein products are indistinguishable from bona fide receptor with respect to sedimentation and electrophoretic mobility, antibody reactivity, and hormone and DNA binding. Moreover, the cloned receptor protein activates its corresponding enhancers, restoring to the receptor-deficient cells the full capacity for regulated enhancement.

Amino Acid Sequence↗

PubMed2003

Identification of two novel RECQL4exonic SNPs and genomic characterization of the IVS12 minisatellite.

Rothmund-Thomson syndrome is a rare autosomal recessive disorder characterized by a widely heterogeneous clinical presentation. Only a subset of clinically diagnosed patients carry RECQL4 gene mutations, probably because of their genetic heterogeneity and/or the complexity of molecular testing. We here describe the polymorphic sites of the RECQL4 gene that detail its genomic structure and may be of interest as modulators of the splicing process and gene expression. We characterized two novel and one already described single-nucleotide polymorphism in the coding region of the RECQL4 gene, which were shown by the exonic splicing enhancer (ESE) score matrix to fall into high-score motifs recognized by serine/arginine-rich proteins. We also describe the genomic structure of a G-C rich minisatellite flanking the 3' splice site of IVS12 in the helicase domain of the RECQL4 gene, which may enhance mutations such as those described at the IVS12 acceptor site. RECQL4 polymorphic sites may be useful for identifying alleles associated with missplicing and, more generally, in cancer-susceptibility association studies.

Adenosine Triphosphatases↗

PubMed2004

Gcn4p and novel upstream activating sequences regulate targets of the unfolded protein response.

Eukaryotic cells respond to accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), a signal transduction pathway that communicates between the ER and the nucleus. In yeast, a large set of UPR target genes has been experimentally determined, but the previously characterized unfolded protein response element (UPRE), an upstream activating sequence (UAS) found in the promoter of the UPR target gene KAR2, cannot account for the transcriptional regulation of most genes in this set. To address this puzzle, we analyzed the promoters of UPR target genes computationally, identifying as candidate UASs short sequences that are statistically overrepresented. We tested the most promising of these candidate UASs for biological activity, and identified two novel UPREs, which are necessary and sufficient for UPR activation of promoters. A genetic screen for activators of the novel motifs revealed that the transcription factor Gcn4p plays an essential and previously unrecognized role in the UPR: Gcn4p and its activator Gcn2p are required for induction of a majority of UPR target genes during ER stress. Both Hac1p and Gcn4p bind target gene promoters to stimulate transcriptional induction. Regulation of Gcn4p levels in response to changing physiological conditions may function as an additional means to modulate the UPR. The discovery of a role for Gcn4p in the yeast UPR reveals an additional level of complexity and demonstrates a surprising conservation of the signaling circuit between yeast and metazoan cells.

Amino Acid Motifs↗

PubMed1997

Expression of the murine CD21 gene is regulated by promoter and intronic sequences.

Murine CD21 gene products are expressed primarily on the surface of B lymphocytes and follicular dendritic cells. To identify the genetic elements that control the tissue-specific expression of the CD21 gene, we analyzed, via transient transfections, the 5' proximal promoter region of the CD21 gene (1272 bp 5' of the initiating ATG). This region possessed strong promoter activity, but it was not tissue specific, in that T cell expression was equivalent to that of B cells. These data suggested that the anticipated tissue-specific control element(s) lies 3' of the initiating ATG. Analysis of a novel minigene construct that possessed both the 5' promoter region and a large region (9 kb) of the CD21 gene 3' of the initiating ATG demonstrated the expected tissue-specific expression. Further analysis using the luciferase reporter system indicated that such control elements reside in the first intron (5.5 kb in size), which separates the exons encoding the signal sequence and the first extracellular short consensus repeat domain of the mature protein. Further dissection of intron 1 demonstrated that the sequences controlling the tissue-specific expression of the murine CD21 gene are contained in the 5' 1.6-kb region of this intron. This 1.6-kb fragment was fractionated into an 800-bp sequence at the 5' end that showed very significant inhibitory activity in both B and T cells and a 3' 800-bp sequence that demonstrated moderate repression in T cells, but enhancer activity in B cells. These data suggest this region of the CD21 gene possesses a number of functionally distinct sites that positively and negatively regulate CD21 gene transcription.

Animals↗

PubMed2002

Pax6 is required for establishing naso-temporal and dorsal characteristics of the optic vesicle.

The establishment of polarity is an important step during organ development. We assign a function for the paired and homeodomain transcription factor Pax6 in axis formation in the retina. Pax6 is a key factor of the highly conserved genetic network implicated in directing the initial phases of eye development. We recently demonstrated that Pax6 is also essential for later aspects of eye development, such as lens formation and retinogenesis. In this study, we present evidence that a highly conserved intronic enhancer, alpha, in the Pax6 gene is essential for the establishment of a distal(high)-proximal(low) gradient of Pax6 activity in the retina. In the mature retina, the activity mediated by the alpha-enhancer defines a population of retinal ganglion cells that project to two sickle-shaped domains in the superior colliculus and lateral geniculate nucleus. Deletion of the alpha-enhancer in vivo revealed that retinal Pax6 expression is regulated in two complementary topographic domains. We found that Pax6 activity is required for the establishment, as well as the maintenance of dorsal and nasotemporal characteristics in the optic vesicle and, later, the optic cup.

Animals↗

PubMed2006

APETALA1 and SEPALLATA3 interact with SEUSS to mediate transcription repression during flower development.

The transcriptional repression of key regulatory genes is crucial for plant and animal development. Previously, we identified and isolated two Arabidopsis transcription co-repressors LEUNIG (LUG) and SEUSS (SEU) that function together in a putative co-repressor complex to prevent ectopic AGAMOUS (AG) transcription in flowers. Because neither LUG nor SEU possesses a recognizable DNA-binding motif, how they are tethered to specific target promoters remains unknown. Using the yeast two-hybrid assay and a co-immunoprecipitation assay, we showed that APETALA1 (AP1) and SEPALLATA3 (SEP3), both MADS box DNA-binding proteins, interacted with SEU. The AP1-SEU protein-protein interaction was supported by synergistic genetic interactions between ap1 and seu mutations. The role of SEU proteins in bridging the interaction between AP1/SEP3 and LUG to repress target gene transcription was further demonstrated in yeast and plant cells, providing important mechanistic insights into co-repressor function in plants. Furthermore, a direct in vivo association of SEU proteins with the AG cis-regulatory element was shown by chromatin immunoprecipitation. Accordingly, a reporter gene driven by the AG cis-element was able to respond to AP1- and SEP3-mediated transcriptional repression in a transient plant cell system when supplied with SEU and LUG. These results suggest that AP1 and SEP3 may serve as the DNA-binding partners of SEU/LUG. Our demonstration of the direct physical interaction between SEU and the C-terminal domain of SEP3 and AP1 suggests that AP1 and SEP3 MADS box proteins may interact with positive, as well as negative, regulatory proteins via their C-terminal domains, to either stimulate or repress their regulatory targets.

Arabidopsis Proteins↗

PubMed2000

[Genetic polymorphism analysis of apoE intron 1 enhancer in patients with coronary heart disease and the association with serum lipid level].

OBJECTIVE: To investigate the association between the polymorphism of apolipoprotein E (apoE) intron 1 enhancer (IE1) and the level of serum lipid in patients with coronary heart disease (CHD). METHODS: ApoE IE1 genotypes of 81 patients with CHD and 108 healthy subjects were detected by polymerase chain reaction and restriction fragment length polymorphism. RESULTS: The frequency of G/G genotype in patients with CHD was significantly higher than that in control group (P < 0.05). The frequency of G allele was significantly higher in patients than in control group (P < 0.05). The gene polymorphism of apoE IE1 obviously influenced the serum lipid levels. The concentrations of TC, and LDL-C among different genotype groups were significantly different, and the level in G/G group was higher than that in C/C group (P < 0.05). CONCLUSION: ApoE IE1 gene polymorphism is closely related to serum lipid, and G/G genotype of apoE IE1 may be a risk factor of coronary heart disease in Han Chinese.

Aged↗