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Results for “Enhancer Elements, Genetic”

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PubMed2006

Multiple cis-acting sequences mediate upregulation of the MDR1 efflux pump in a fluconazole-resistant clinical Candida albicans isolate.

Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the antimycotic agent fluconazole and other metabolic inhibitors in clinical Candida albicans strains. Constitutive MDR1 overexpression in such strains is caused by mutations in as yet unknown trans-regulatory factors. In order to identify the cis-acting sequences in the MDR1 regulatory region that mediate constitutive MDR1 upregulation, we performed a promoter deletion analysis in the genetic background of an MDR1-overexpressing clinical C. albicans isolate. We found that several different regions in the MDR1 promoter can mediate MDR1 overexpression in this isolate. In contrast, deletion of one of these regions abolished benomyl-induced MDR1 expression in a C. albicans laboratory strain. These results suggest that multiple transcription factors control expression of the MDR1 efflux pump in C. albicans and that the mutation(s) that causes constitutive MDR1 overexpression and drug resistance in clinical C. albicans isolates affects the activities of several of these transcription factors.

Antifungal Agents↗

PubMed2001

Activation of a fibroblast-specific enhancer of the proalpha2(I) collagen gene in tight-skin mice.

OBJECTIVE: Reporter transgenes were introduced into the type 1 tight-skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast-specific genetic programs are activated in fibrosis. METHODS: Transgenes harboring upstream fragments of the 5' flanking region of the mouse proalpha2(I) collagen gene (Col1a2), linked to a 400-bp minimal Col1a2 promoter driving an Escherichia coli beta-galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding. Expression of these transgenes, which function as lineage-specific markers of fibroblast differentiation, was compared between the Tsk-LacZ mice and non-Tsk littermates. Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor beta1 (TGFbeta1), was investigated by transient transfection of reporter constructs in tissue-culture cells. RESULTS: There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age. This was maximal at 6 weeks old (mean +/- SD 237 +/- 24% of non-Tsk controls; P= 0.001). Recombinant TGFbeta1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk-LacZ fibroblasts showed elevated LacZ expression in tissue culture. CONCLUSION: These data suggest that activating signals in Tsk1/+ mice may act via fibroblast-specific regulatory elements within the murine Col1a2 gene. Although TGFbeta has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFbeta responsive in vitro, our results suggest that fibroblast-specific pathways may also be involved.

Animals↗

PubMed2004

A zinc finger transcription factor, ZicL, is a direct activator of Brachyury in the notochord specification of Ciona intestinalis.

In ascidian embryos, Brachyury is expressed exclusively in blastomeres of the notochord lineage and play an essential role in the notochord cell differentiation. The genetic cascade leading to the transcriptional activation of Brachyury in A-line notochord cells of Ciona embryos begins with maternally provided beta-catenin, which is essential for endodermal cell specification. beta-catenin directly activates zygotic expression of a forkhead transcription factor gene, FoxD, at the 16-cell stage, which in turn somehow activates a zinc finger transcription factor gene, ZicL, at the 32-cell stage, and then Brachyury at the 64-cell stage. One of the key questions to be answered is whether ZicL functions as a direct activator of Brachyury transcription, and this was addressed in the present study. A fusion protein was constructed in which a zinc finger domain of Ciona ZicL was connected to the C-terminus of GST. Extensive series of PCR-assisted binding site selection assays and electrophoretic mobility shift assays demonstrated that the most plausible recognition sequence of Ciona ZicL was CCCGCTGTG. We found the elements CACAGCTGG (complementary sequence: CCAGCTGTG) at -123 and CCAGCTGTG at -168 bp upstream of the putative transcription start site of Ci-Bra in a previously identified basal enhancer of this gene. In vitro binding assays indicated that the ZicL fusion protein binds to these elements efficiently. A fusion gene construct in which lacZ was fused with the upstream sequence of Ci-Bra showed the reporter gene expression exclusively in notochord cells when the construct was introduced into fertilized eggs. In contrast, fusion constructs with mutated ZicL-binding-elements failed to show the reporter expression. In addition, suppression of Ci-ZicL abolished the reporter gene expression, while ectopic and/or overexpression of Ci-ZicL resulted in ectopic reporter expression in non-notochord cells. These results provide evidence that ZicL directly activates Brachyury, leading to specification and subsequent differentiation of notochord cells.

Amino Acid Sequence↗

PubMed1997

CNS midline cells in Drosophila induce the differentiation of lateral neural cells.

Cells located at the midline of the developing central nervous system perform a number of conserved functions during the establishment of the lateral CNS. The midline cells of the Drosophila CNS were previously shown to be required for correct pattern formation in the ventral ectoderm and for the induction of specific mesodermal cells. Here we investigated whether the midline cells are required for the correct development of lateral CNS cells as well. Embryos that lack midline cells through genetic ablation show a 15% reduction in the number of cortical CNS cells. A similar thinning of the ventral nerve cord can be observed following mechanical ablation of the midline cells. We have identified a number of specific neuronal and glial cell markers that are reduced in CNS midline-less embryos (in single-minded embryos, in early heat-shocked Notch(ts1) embryos or in embryos where we mechanically ablated the midline cells). Genetic data suggest that both neuronal and glial midline cell lineages are required for differentiation of lateral CNS cells. We could rescue the lateral CNS phenotype of single-minded mutant embryos by transplantation of midline cells as well as by homotopic expression of single-minded, the master gene for midline development. Furthermore, ectopic midline cells are able to induce enhanced expression of some lateral CNS cell markers. We thus conclude that the CNS midline plays an important role in the differentiation or maintenance of the lateral CNS cortex.

Animals↗

PubMed1992

Efficient replication and expression of murine leukemia virus with major deletions in the enhancer region of U3.

The effect of deletions within the enhancer region in the U3 part of the LTR derived from the murine retrovirus Akv was studied. The deletions were stably transmitted through normal virus replication as shown by sequence analysis of cloned polymerase chain reaction product of the cDNA copy of the viral RNA. Genetic tagging of the retrovirus with lacO facilitated the analysis. Among the individual mutated LTRs an over 100-fold difference in a transient expression assay was previously detected. This difference was not revealed in studies of viral replication in cell culture, where the expression level of virus with the deleted LTRs all reached the level of virus with the intact LTR. We propose that stimulatory cis-acting sequences either adjacent to the site of proviral integration or in the coding regions of the provirus may compensate for deletions in the LTR.

3T3 Cells↗

PubMed2000

Demystified.... Transcription.

The transcription of genes could be defined as the intricate molecular manoeuvres occurring in the nuclei of cells, which allow the translation of genetic information held in the DNA into the proteins required for life. Gene transcription is the dominant control point in the production of any protein, and is initiated and regulated through the combined activities of a highly specialised set of nuclear proteins. This review examines the role of these protein "transcription factors" in the production of messenger RNA, the information intermediary produced in the nucleus, and transferred to the cytoplasm to serve as a template for protein synthesis. In combination with RNA polymerase, an extraordinary and complex enzyme required to synthesise new RNA molecules, a multitude of transcription factors combine their activities to orchestrate and control this elegant process.

Enhancer Elements, Genetic↗

PubMed2025

Recent evolution of the developing human intestine affects metabolic and barrier functions.

Diet, microbiota, and other exposures make the intestinal epithelium a nexus for evolutionary change; however, little is known about genomic changes associated with adaptation to a distinctly human environment. In this work, we interrogate the evolution of cell types in the developing human intestine by comparing tissue and organoids from humans, chimpanzees, and mice. We find that recent changes in primates are associated with immune barrier function and lipid and xenobiotic metabolism and that human-specific genetic features affect these functions. Enhancer assays, genetic deletion, and in silico mutagenesis resolve evolutionarily important enhancers of lactase (LCT) and insulin-like growth factor binding protein 2 (IGFBP2). Altogether, we identify the developing human intestinal epithelium as a rapidly evolving system and show that great ape organoids provide insight into human biology.

Animals↗

PubMed2002

Cardiogenesis in the Drosophila model: control mechanisms during early induction and diversification of cardiac progenitors.

The dorsal vessel of Drosophila displays developmental, functional, and morphological similarities to the primitive linear heart tube of early vertebrate embryos. Because these similarities extend to the genetic and molecular level, Drosophila has become a fruitful model to study control mechanisms of early heart development. Herein we summarize recently obtained insights into control mechanisms during early induction and diversification of cardiac progenitors in Drosophila. We also show that induction of tinman, a key cardiogenic gene, in the dorsal mesoderm by Dpp (Drosophila BMP) involves protein/protein interactions between Tinman and the Smad proteins Mad and Medea, in addition to their DNA-binding activities to specific tinman enhancer sequences. Furthermore, we present evidence that binding of a high-mobility-group protein, HMG-D, to the Dpp-responsive enhancer of tinman as well as to the Tinman protein may be involved in the formation of a fully active enhancer complex.

Animals↗

PubMed2004

Polymorphisms in TDG and MGMT genes - epidemiological and functional study in lung cancer patients from Poland.

Functional genetic polymorphisms of DNA repair genes are good candidates for cancer susceptibility markers. We studied two genes coding for proteins removing small DNA adducts by direct repair (MGMT), or mispaired DNA bases by base excision repair (TDG). The non-silent polymorphisms of MGMT (84:Phe, 143:Val, 178:Arg) and TDG (199:Ser, 367:Met), and the functional MGMT enhancer polymorphism, did not show any statistically significant association with lung cancer risk in our case-control analysis, but due to the relatively small number of individuals, strong conclusions on cancer risk association or lack thereof cannot be made. Sequencing of the TDG cDNA has not revealed any novel polymorphism, but did find an alternatively spliced mRNA missing exon 2. Our search for polymorphisms within the promoter-enhancer region of MGMT revealed three novel sequence variants. The functional significance of the previously published MGMT enhancer polymorphism (1099C->T) was assessed. The less frequent sequence variant of the enhancer was associated with a modest (16-64%), but statistically significant, increase of MGMT promoter-enhancer activity in the studied cell lines. This work points to the importance of studying the expression-regulating elements of genes, as they may contain functional polymorphisms with the potential for modulating risk of various diseases, including cancer.

Adult↗

PubMed2006

Autism and ultraconserved non-coding sequence on chromosome 7q.

OBJECTIVE: Autism has been linked to a broad region on chromosome 7q that contains a large number of genes involved in transcription and development. This region is also enriched for ultraconserved non-coding elements, defined as human-rodent sequences that are 100% aligned over > or =200 base pairs, which have a high likelihood of being functional. Therefore, as only a few rare coding variants have been detected in the autism candidate genes on 7q examined to date, we decided to screen these ultraconserved elements for possible autism susceptibility alleles. METHODS: We used denaturing high-performance liquid chromatography, and DNA sequencing, to perform variant detection in a total of 146 cases with autism, 96 from the Autism Genetic Resource Exchange and 50 from the Central Valley of Costa Rica, as well as 124 controls from the Polymorphism Discovery Resource Panel. We screened 10 consecutive ultraconserved elements in, or flanking, the genes DLX5/6, AUTS2 and FOXP2 on chromosome 7q. RESULTS: Although we did find several rare variants in autism cases that were not present in controls, we also observed rare variants present in controls and not cases. The most common variant occurred in controls at a frequency of 3.3%. Interestingly, two ultraconserved elements each harbored three independent variants and one ultraconserved element harbored two independent variants, suggesting that ultraconservation is maintained chiefly by a decreased tendency toward fixation, rather than a significantly lower mutation rate. CONCLUSIONS: Our results show that these sequences are unlikely to harbor major autism susceptibility alleles.

Autistic Disorder↗

PubMed2004

Identification of a critical control element directing expression of the muscle-specific transcription factor MRF4 in the mouse embryo.

Skeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between -7.5 and -3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between -6.6 and -5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (-58/-48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer.

Animals↗

PubMed2003

Comparison of hCMV immediate early and CaMV 35S promoters in both plant and human cells.

Cauliflower mosaic virus 35S promoter, widely used in transgenic crop plants, is known to be recognized in widely differing kinds of cells. Its activity in human cells may have impact on the risk assessment for the environmental release of genetically modified plants. In this study, transient expression of several constructs containing beta-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells. The results showed very low but measurable activity of 35S promoter in human 293T-cells (0.01% of that revealed when using pCMV) and in 293 cells that do not produce SV40 T antigen this activity was even lower. On the other hand, in potato protoplasts, pCMV displayed nearly 1% activity seen with p35S.

Bacterial Outer Membrane Proteins↗

PubMed2026

Exhausted CD8+ T cell fate is programmed by dynamic CTCF-mediated enhancer activation and invariant CTCF-imposed barriers.

Exhausted CD8+ T (TEX) cells undergo extensive genome reorganization during differentiation, yet the drivers of this process remain elusive. Here we show that CTCF programmed CD8+ TEX cell fates through two distinct modes of action. CTCF acquired de novo binding sites and concordantly induced open chromatin in early CD8+ TEX cells responding to chronic viral infection. The dynamic CTCF binding activated enhancers and promoted chromatin looping. Consequently, genetic ablation of CTCF diminished chromatin accessibility and interaction strength, impairing CD8+ TEX cell proliferation, effector function and bioenergetic mobilization. Conversely, invariant CTCF binding acted as essential chromatin barriers, and loss of CTCF disrupted insulation and caused aberrant chromatin self-association and undue RNA polymerase II pausing, leading to excessive activation of exhaustion- and stemness-linked genes. Thus, CTCF balanced CD8+ TEX cell differentiation by gaining dynamic binding to induce cytotoxicity and sustain metabolic fitness, while its invariant binding compartmentalized exhaustion and stemness program genes to prevent their overexuberant activation.

CCCTC-Binding Factor↗

PubMed1987

Regulation of inducible and tissue-specific gene expression.

Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.

Animals↗

PubMed2002

The Drosophila slamdance gene: a mutation in an aminopeptidase can cause seizure, paralysis and neuronal failure.

We report here the characterization of slamdance (sda), a Drosophila melanogaster "bang-sensitive" (BS) paralytic mutant. This mutant exhibits hyperactive behavior and paralysis following a mechanical "bang" or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because it has a drastically lowered seizure threshold. Through genetic mapping, molecular cloning, and RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Furthermore, using mRNA in situ hybridization and LacZ staining, we have found that the sda gene is expressed specifically in the central nervous system at particular developmental stages. Together, these results suggest that the bang sensitivity in sda mutants is caused by a defective APN gene that somehow increases seizure susceptibility. Finally, by using the sda mutation as a sensitized background, we have been able to identify a rich variety of sda enhancers and other independent BS mutations.

Amino Acid Sequence↗

PubMed1997

Analysis of P[gal4] insertion lines of Drosophila melanogaster as a route to identifying genes important in the follicle cells during oogenesis.

We report the analysis of a number of lines of Drosophila melanogaster containing insertions of the yeast gal4 gene. By crossing a UAS-lacZ fusion gene as a reporter into these lines, we analysed the expression patterns of beta-galactosidase during oogenesis. Since there is no expression of GAL4 in the germ-line in these experiments, this is an ideal system for the analysis of expression patterns in sub-sets of follicle cells. These lines provide ideal markers for sets of follicle cells, e.g. anterior or posterior polar cells for studying genetic interactions in oogenesis; however, they can also be used in the same way as conventional enhancer traps to clone nearby genes with similar expression patterns. The advantages of this dual gal4/UAS system over conventional enhancer trapping includes the possibility of GAL4-directed misexpression and antisense expression studies to establish the function of the genes we identified during follicle cell determination and differentiation. These studies could lead to the isolation of homologous genes crucial in mammalian oogenesis. Understanding how the somatic cells and germ cells interact to promote growth and maturation of the mammalian follicle and oocyte could well be crucial for improving the fertility of eggs used for in-vitro fertilization programmes, and could provide methods for assessing the quality of eggs.

Animals↗

PubMed1999

Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A.

The transcription factors EBF and E2A are required at a similar step in early B cell differentiation. EBF and E2A synergistically upregulate transcription of endogenous B cell-specific genes in a non-B cell line. Here, we examine a genetic collaboration between these factors in regulating B lymphopoiesis. We find that Ebf+/- E2a+/- mice display a marked defect in pro-B cell differentiation at a stage later than observed in the single homozygous mutant mice. Pro-B cells from Ebf+/- E2a+/- mice show reduced expression of lymphoid-specific transcripts, including Pax5, Rag1, Rag2, and mb-1. We also show that EBF directly binds and activates the Pax5 promoter. Together, these data show collaboration between EBF and E2A and provide insight into the hierarchy of transcription factors that regulate B lymphocyte differentiation.

Animals↗

PubMed1995

Variation of human papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world.

We examined the genomic diversity of human papillomavirus type 6 (HPV-6) and HPV-11 isolates from different parts of the world by comparing the nucleotide sequences of part of the long control region of three reference clones and 62 HPV-6 and 40 HPV-11 isolates from Africa, Europe, Asia, and North and South America. The genomic sequence of the HPV-6b reference type had to be amended by inclusion of a 94-bp segment, which is also present with minor differences in HPV-6a. Aside from two small inserts typical of all variants related to HPV-6a and three inserts found in HPV-11 variants, no major alterations to the size of the long control regions of these viruses were observed. This corrects the previous impression that these two HPV types are highly polymorphic. Altogether, 19 HPV-6 and 10 HPV-11 variant genomes could be distinguished, and most of the differences were due to point substitutions. The variants of either type were continuously connected in phylogenetic trees rather than clustered separately into subtype groups. Thirteen mutations, namely, the two HPV-6a inserts and 11 substitutions in HPV-6 or HPV-11 variants, reduced the dissimilarity between the types, but they bridged only a small fraction of the genetic distance between the two types. Genomes more obviously intermediate between HPV-6 and HPV-11 were not found and probably do not exist any more. A single HPV-11 variant was found in Africa, but otherwise, no significant correlations of HPV-6 or HPV-11 variants with geography or ethnicity of the patient cohort were found. Functional analysis of diverse enhancer variants showed activities that differed two-to threefold, and it must be considered that transcriptional differences may alter the biology or pathology of these viruses. Similar variants were found in lesions from anatomically different sites and in both benign and malignant lesions.

Base Sequence↗