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Results for “Enhancer Elements, Genetic”

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PubMed1991

The distribution of transcripts of neurogenic genes in neurogenic mutants of Drosophila melanogaster.

The neurogenic genes of Drosophila melanogaster are required for correct separation of neural and epidermal progenitor cells during early embryogenesis. Results from genetic analyses indicate that the neurogenic genes are functionally related. We have studied the spatial distribution of RNA from the neurogenic genes D1, neu, and m4, m5, m7 and E(spl) [four genes of the Enhancer of split complex] in various neurogenic mutant embryos by in situ hybridization. An abnormal distribution of RNA from certain of the genes is found in neurogenic mutants, suggesting that at least some of the functional interactions inferred from genetic data take place at the transcriptional level. We discuss these results in relation to the events of early neurogenesis.

Animals↗

PubMed1997

Concomitant downregulation of IgH 3' enhancer activity and c-myc expression in a plasmacytoma x fibroblast environment: implications for dysregulation of translocated c-myc.

Regulation of immunoglobulin heavy chain (IgH) gene expression is controlled by a B cell-specific promoter, intronic enhancer and additional B cell-specific enhancer elements identified recently in the 3' end of the IgH locus. One of the latter elements, the IgH 3' enhancer, is of particular interest: (1) it is B cell-specific and active only in late B cell development; (2) in rodent plasmacytomas and in some human Burkitt's lymphomas it is part of a locus control region (LCR) that is involved in deregulation of the c-myc oncogene as a result of translocation into the IgH locus; and (3) it has been implicated in the mechanisms that control Ig gene class switch recombination. We have used a somatic cell hybridization approach to genetically analyse regulation of the activity of the IgH 3' enhancer. When mouse MPC11 plasmacytoma cells, in which the IgH 3' enhancer is active, are fused with fibroblasts, Ig expression is extinguished at the level of transcription. Here we show that in a MPC11 plasmacytoma x fibroblast environment, the IgH 3' enhancer is transcriptionally inactive. Furthermore, we demonstrate that binding of several B cell-specific transcription factors, essential for IgH 3' enhancer activity, is lacking, which may explain 3' enhancer inactivity, although the binding of repressors cannot be excluded. Moreover, the high expression level of c-myc, characteristic of the parental MPC11 cells carrying the t(12;15) translocation, is down-regulated in the hybrids to that in unfused fibroblasts. Therefore, inactivation of the IgH 3' enhancer is a multifactorial process affecting several transcription factors that control the cell-specific and developmental activity of the enhancer.

Animals↗

PubMed2000

A tyramine receptor gene mutation causes a defective olfactory behavior in Drosophila melanogaster.

We characterized molecular profiles of a new olfactory mutant line, honoka (hono), which was found among 500 viable P-element insertion lines screened first by 5-bromo-4-chloro-3-indrolyl-beta-D-galactopyranoside (X-gal) staining on the third segment of the antenna, and then by behavioral assays to several pure chemicals. The behavioral responses of hono mutants to repellents such as ethyl acetate (EA), benzaldehyde (BZ) and 4-methylcycrohexanol (MCH), were reduced compared with those of a control strain. The location of the P-element insertion was determined to be about 100bp) upstream of the first exon of the tyramine receptor gene. The level of 3.6kb tyramine receptor mRNA expression was reduced in hono compared with that of wild-type flies. The tyramine receptor cDNA hybridized to the chromosomal division 79C-D, the same locus as the P-element insertion point in hono, and not to 99A-B, previously reported by Arakawa et al. (1990. Neuron 2, 343-354). Electrophysiological responses to octopamine and tyramine were examined by measuring the excitatory junctional potential (EJP) amplitude from larval body-wall muscles of the hono mutant. The hono was impaired with responding to tyramine, while displaying normal response to octopamine. These results indicate that tyramine has a functional role in the Drosophila olfactory system as a neurotransmitter or a neuromodulator, and hono is the first tyramine receptor mutant. This study provides the first step toward understanding of the molecular genetics of tyramine-mediated neural functions in Drosophila.

Acetates↗

PubMed1994

Epigenetic lesions at the H19 locus in Wilms' tumour patients.

To test the potential role of H19 as a tumour suppressor gene we have examined its expression and DNA methylation in Wilms' tumours (WTs). In most WTs (18/25), H19 RNA was reduced at least 20-fold from fetal kidney levels. Of the expression-negative tumours ten retained 11p15.5 heterozygosity: in nine of these, H19 DNA was biallelically hypermethylated and in two cases hypermethylation locally restricted to H19 sequences was also present in the non-neoplastic kidney parenchyma. IGF2 mRNA was expressed in most but not all WTs and expression patterns were consistent with IGF2/H19 enhancer competition without obligate inverse coupling. These observations implicate genetic and epigenetic inactivation of H19 in Wilms' tumorigenesis.

Alleles↗

PubMed2025

The common murine retroviral integration site activating Hhex marks a distal regulatory enhancer co-opted in human early T-cell precursor leukemia.

The Hhex gene encodes a transcription factor that is important for both embryonic and post-natal development, especially of hematopoietic tissues. Hhex is one of the most common sites of retroviral integration in mouse models. We found the most common integrations in AKXD (recombinant inbred strains) T-ALLs occur 57-61kb 3' of Hhex and activate Hhex gene expression. The genomic region of murine leukemia virus (MLV) integrations has features of a developmental stage-specific cis regulatory element (CRE), as evidenced by ATAC-seq in murine progenitor cells and high H3K27 acetylation at the syntenic CRE in human hematopoietic cell lines. With ChIP-exonuclease, we describe occupancy of LIM domain binding protein 1 (LDB1), the constitutive partner of the LIM Only-2 (LMO2), GATA1, and TAL1 transcription factors at GATA sites and at a composite GATA-E box within the CRE. With virtual 4C analysis, we observed looping between this +65kb CRE and the proximal intron one enhancer of HHEX in primary human ETP-ALLs and in normal progenitor cells. Our results show that retroviral integrations at intergenic sites can mark and take advantage of CREs. Specifically, in the case of HHEX activation, this newly described +65kb CRE is co-opted in the pathogenesis of ETP-ALL by the LMO2/LDB1 complex.

Humans↗

PubMed2001

Identification of chromosomal deficiencies that modify Drosophila mastermind mutant phenotypes.

Mastermind (Mam) is a component of Notch pathway signaling. In combination with the intracellular domain of Notch and Suppressor of Hairless, Mam forms a transcriptional activation complex. We have initiated a genetic approach to identify other loci involved in Mam function. The screen utilizes engineered mutations in Mam that derive from GAL4-UAS-directed expression of dominant negative constructs. When driven at the wing margin, truncated versions of Mam phenocopy Notch pathway mutations. Correlated with these phenotypes is depression of Notch pathway target expression. Strains expressing truncated versions of Mam were tested for genetic interactions with a large collection of chromosomal deficiencies. Genomic segments that enhanced and suppressed the dominant wing phenotype were identified. These regions may contain uncharacterized loci involved in Notch pathway function.

Animals↗

PubMed2004

Regulation of GATA gene expression during vertebrate development.

GATA factors regulate critical events in hematopoietic lineages (GATA-1/2/3), the heart and gut (GATA-4/5/6) and various other tissues. Transgenic approaches have revealed that GATA genes are regulated in a modular fashion by sets of enhancers that govern distinct temporal and/or spatial facets of the overall expression patterns. Efforts are underway to resolve how these GATA gene enhancers are themselves regulated in order to elucidate the genetic and molecular hierarchies that govern GATA expression in particular developmental contexts. These enhancers also afford a raft of tools that can be used to selectively perturb and probe various developmental events in transgenic animals.

Animals↗

PubMed2001

Pannier is a transcriptional target and partner of Tinman during Drosophila cardiogenesis.

During Drosophila embryogenesis, the homeobox gene tinman is expressed in the dorsal mesoderm where it functions in the specification of precursor cells of the heart, visceral, and dorsal body wall muscles. The GATA factor gene pannier is similarly expressed in the dorsal-most part of the mesoderm where it is required for the formation of the cardial cell lineage. Despite these overlapping expression and functional properties, potential genetic and molecular interactions between the two genes remain largely unexplored. Here, we show that pannier is a direct transcriptional target of Tinman in the heart-forming region. The resulting coexpression of the two factors allows them to function combinatorially in the regulation of cardiac gene expression, and a physical interaction of the proteins has been demonstrated in cultured cells. We also define functional domains of Tinman and Pannier that are required for their synergistic activation of the D-mef2 differentiation gene in vivo. Together, these results provide important insights into the genetic mechanisms controlling heart formation in the Drosophila model system.

Animals↗

PubMed2006

Identification of genes that interact with Drosophila liquid facets.

We have performed mutagenesis screens of the Drosophila X chromosome and the autosomes for dominant enhancers of the rough eye resulting from overexpression of liquid facets. The liquid facets gene encodes the homolog of vertebrate endocytic Epsin, an endocytic adapter protein. In Drosophila, Liquid facets is a core component of the Notch signaling pathway required in the signaling cells for ligand endocytosis and signaling. Why ligand internalization by the signaling cells is essential for signaling is a mystery. The requirement for Liquid facets is a hint at the answer, and the genes identified in this screen provide further clues. Mutant alleles of clathrin heavy chain, Rala, split ends, and auxilin were identified as enhancers. We describe the mutant alleles and mutant phenotypes of Rala and aux. We discuss the relevance of all of these genetic interactions to the function of Liquid facets in Notch signaling.

Animals↗

PubMed1990

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.

Animals↗

PubMed2006

Genetic visualization of neurogenesis.

Neurons are generated from stem or progenitor cells in discrete areas in the adult brain. The exact temporal and spatial distribution of adult neurogenesis has, however, been difficult to establish because of inherent limitations with the currently used techniques, and there are numerous controversies with regard to whether neurons are generated in specific regions or in response to insults. We describe here the generation of transgenic mice that express conditionally active Cre recombinase under the control of a nestin enhancer element. These mice allow the recombination of reporter alleles specifically in neural stem and progenitor cells and the visualization of their progeny in the adult brain. This offers a simple and efficient way to visualize live adult born neurons without the caveats of currently used techniques.

Animals↗

PubMed2004

Transforming growth factor-beta1 genotype in sporadic breast cancer patients from India: status of enhancer, promoter, 5'-untranslated-region and exon-1 polymorphisms.

Transforming growth factor beta (TGF-beta) is an example for a large and still-growing family of growth factors. TGF-beta1 is known to act both as a tumour suppressor and as a stimulator of tumour progression. This study examines the relationship amongst putative enhancer, promoter, 5'-untranslated-region (UTR) and exon-1 polymorphisms of the TGF-beta1 gene (region I from -1881 to -1613; region II from -1410 to -1123, and region III from -55 to +176, as per human genome organisation (HUGO) nomenclature) in 26 breast cancer patients and 97 healthy control subjects. The germline and somatic status of the four known polymorphisms was ascertained, and a significant difference was observed for the germline C/T and T/T genotype distribution between patients and controls in comparison to C/C genotypes at position -1349 (chi2 = 6.193; P = 0.009). In addition to the somatic variations observed for some of the regions studied, in 10/26 (38%) sporadic breast cancer cases, a novel somatic mutation in codon 47 of exon 1 (GenBank accession number AY059373) was also detected in tumour samples. The risk of cancer was found to be significant (OR = 4.525) for the -1349 C/T and T/T genotype background, suggesting that this genetic background may act as a risk factor for sporadic breast cancer.

5' Untranslated Regions↗

PubMed1987

Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses.

Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.

Animals↗

PubMed1994

Identification and characterization of multiple A/T-rich cis-acting elements that control expression from Dictyostelium actin promoters: the Dictyostelium actin upstream activating sequence confers growth phase expression and has enhancer-like properties.

The promoter elements in the Dictyostelium actin 15 and actin 6 genes required for full growth phase expression were identified by assaying promoter/luciferase reporter constructs. We find that these promoters contain common cis-acting elements, an actin upstream activating sequence (UAS) and sequences proximal to the transcription start site that overlap with a poly(dT) region. The actin 15 promoter has two additional cis-acting elements not present in the actin 6 promoter that may account for the higher level of expression from the actin 15 promoter. All of the identified promoter elements are unusual for Dictyostelium in that they are all A/T-rich. Two cis-acting elements, the actin UAS and the poly(dT) domain were studied in greater detail. The actin UAS was tested on a heterologous promoter from the prespore-specific gene SP60 and shown to have the ability to confer growth phase expression. The actin UAS also exhibited the ability to function in a distance- and orientation-independent manner and activate expression synergistically when present in two copies. The poly(dT) domain of the actin 15 promoter was studied in greater detail by using a genetic selection scheme to define parameters that effect the strength of this element. This element is comprised of 45 consecutive dT residues immediately upstream of the putative TATA box. We show that the length of the homopolymer dT region correlates with the expression level of the promoter. The poly(dT) element is also shown to function to promote wild-type levels of expression with small deviations in the sequence, indicating that the element is not required to be homopolymeric to function.

Actins↗

PubMed2001

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.

Animals↗

PubMed2004

An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster.

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.

Animals↗

PubMed2002

Glucose-regulated glucose uptake by transplanted muscle cells expressing glucokinase counteracts diabetic hyperglycemia.

Type 1 diabetic patients depend on insulin replacement therapy. However, chronic hyperglycemia due to failure to maintain proper glycemic control leads to microvascular, macrovascular, and neurological complications. Increased glucose disposal by tissues engineered to overexpress key regulatory genes in glucose transport or phosphorylation can reduce diabetic hyperglycemia. Here we report that differentiated myoblast cells expressing the glucose-phosphorylating enzyme glucokinase (GK) showed a glucose-dependent increase in glucose uptake and utilization in vitro. Transplantation of GK-expressing myotubes into healthy mice did not alter blood glucose levels and recipient mice maintained normoglycemia. After streptozotocin treatment, mice transplanted with GK-expressing myotubes counteracted hyperglycemia, polydipsia, and polyphagia, whereas mice transplanted with control myotubes developed diabetes. Similarly, diabetic mice transplanted with control myotubes remained hyperglycemic. In contrast, transplantation of GK-expressing myotubes into diabetic mice lowered hyperglycemia. These results suggest that the use of genetically engineered muscle cells to express glucokinase may provide a glucose-regulated approach to reduce diabetic hyperglycemia.

Animals↗

PubMed1993

In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor.

Recent genetic experiments have suggested that tat transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat requires functional upstream enhancer sequences--Sp1 sites, in particular. In these experiments, HeLa cell nuclear extracts were passed over affinity matrices containing chemically synthesized or bacterially expressed HIV-1 Tat. Assay of material that bound to and eluted from the Tat matrices revealed the presence of the Sp1 transcription factor. Other transcription factors (Oct and NF-kappa B) also bound to Tat matrices but with less efficiency--in parallel with the lower capacities of these binding motifs to confer Tat responsiveness on a basal HIV-1 promoter compared with Sp1 sites. Passage of nuclear extracts over matrices containing other neutral proteins, including bovine serum albumin, ovalbumin, and lysozyme, revealed no or reduced binding. Cross-linking experiments indicated that the purified Sp1 and Tat proteins can form multimeric complexes in the absence of other proteins. The region of Tat responsible for Sp1 binding was localized to a region encompassing residues 30 to 62. Immunoprecipitation experiments with HIV-1-infected T lymphocytes indicated coimmunoprecipitation of Tat and Sp1. These experiments extend previous genetic experiments and suggest a direct interaction between Tat and Sp1 during transactivation.

Base Sequence↗