Transgenic flies expressing the fluorescence calcium sensor Cameleon 2.1 under UAS control.
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Animals↗
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Results for “Enhancer Elements, Genetic”
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Conditional expression of UAS-transgenes in the adult eye with a new gene-switch vector system.
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Animals↗
The Dorothy enhancer has Tinman binding sites and drives hopscotch-induced tumor formation.
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Animals↗
Multiple roles for notch in Drosophila myogenesis.
Notch plays a role in many cell fate decisions in the developing Drosophila embryo, often at successive stages during the formation of a single tissue. In the embryonic mesoderm, Notch is involved in the process by which muscle progenitors are selected from a field of equivalent myoblasts. We have investigated the roles of Notch in somatic myogenesis and show that Notch can affect at least two additional steps in muscle development. Subsequent to the initial specification of progenitors, myoblast identity remains sensitive to mesodermal Notch activity until the time of fusion. Additionally, Notch is capable of suppressing muscle development nonautonomously by regulating a signal that emanates from the ectoderm.
Animals↗
Genome-wide prediction of mammalian enhancers based on analysis of transcription-factor binding affinity.
Understanding the regulation of human gene expression requires knowledge of the "second genetic code," which consists of the binding specificities of transcription factors (TFs) and the combinatorial code by which TF binding sites are assembled to form tissue-specific enhancer elements. Using a novel high-throughput method, we determined the DNA binding specificities of GLIs 1-3, Tcf4, and c-Ets1, which mediate transcriptional responses to the Hedgehog (Hh), Wnt, and Ras/MAPK signaling pathways. To identify mammalian enhancer elements regulated by these pathways on a genomic scale, we developed a computational tool, enhancer element locator (EEL). We show that EEL can be used to identify Hh and Wnt target genes and to predict activated TFs based on changes in gene expression. Predictions validated in transgenic mouse embryos revealed the presence of multiple tissue-specific enhancers in mouse c-Myc and N-Myc genes, which has implications for organ-specific growth control and tumor-type specificity of oncogenes.
Amino Acid Sequence↗
Positive autoregulation of the Myocyte enhancer factor-2 myogenic control gene during somatic muscle development in Drosophila.
The myocyte enhancer factor-2 (MEF2) transcription factor plays a central role in the activation and maintenance of muscle gene expression in fruit flies and vertebrates. The mechanism of action and downstream target genes of MEF2 have been defined in considerable detail, but relatively little is known about the mechanisms that regulate MEF2 expression during muscle development. Here we demonstrate that MEF2 maintains its own expression in all differentiated muscle cell types during late embryonic and larval development in Drosophila by binding a conserved MEF2 site in a muscle-specific regulatory enhancer. Ectopic expression of Mef2 is sufficient to directly activate this enhancer in some, but not all, non-muscle cells. Furthermore, activation of the Mef2 enhancer normally in muscle cells and ectopically in non-muscle cells is dependent upon the integrity of the MEF2 binding site. These findings suggest an evolutionarily conserved mechanism whereby MEF2 can stabilize the muscle phenotype by sustaining its own expression through a myogenic autoregulatory loop.
Animals↗
Dual functions of E2F-1 in a transgenic mouse model of liver carcinogenesis.
Deregulation of E2F transcriptional control has been implicated in oncogenic transformation. Consistent with this idea, we recently demonstrated that during hepatocarcinogenesis in c-myc/TGFalpha double transgenic mice, there is increased expression of E2F-1 and E2F-2, as well as induction of putative E2F target genes. Therefore, we generated transgenic mice expressing E2F-1 under the control of the albumin enhancer/promoter to test the hypothesis that E2F family members may contribute to liver tumor development. Overexpression of E2F-1 resulted in mild but persistent increases in cell proliferation and death during postnatal liver growth, and no increases in hepatic regenerative growth in response to partial hepatectomy. Nevertheless, from 2 months postnatally E2F-1 transgenic mice exhibited prominent hepatic histological abnormalities including preneoplastic foci adjacent to portal tracts and pericentral large cell dysplasia. From 6 to 8 months onward, there was an abrupt increase in the number of neoplastic nodules ('adenomas') with 100% incidence by 10 months. Some adenomas showed evidence of malignant transformation, and two of six mice killed at 12 months showed trabecular hepatocellular carcinoma. Endogenous c-myc was up-regulated in the early stages of E2F-1 hepatocarcinogenesis, whereas p53 was overexpressed in the tumors, suggesting that both E2F-1-mediated proliferation and apoptosis are operative but at different stages of hepatocarcinogenesis. In conclusion, E2F-1 overexpression in the liver causes dysplasia and tumors and suggests a cooperation between E2F-1 and c-myc oncogenes during liver oncogenesis.
Albumins↗
An endogenous suppressor of hairy-wing insulator separates regulatory domains in Drosophila.
Insulators define independent domains of gene function throughout the genome. The Drosophila gypsy insulator was isolated from the gypsy retrotransposon as a region that contains a cluster of binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. To study the effects of the gypsy insulator on gene expression within a single genomic domain, targeted gene replacement was used to exchange the endogenous yellow gene, located at cytological location 1A, with a set of gypsy-modified yellow genes. Replaced yellow genes carried a gypsy insulator positioned between the yellow promoter and either the upstream or the downstream tissue-specific enhancers. Whereas the gypsy insulator blocked the function of the upstream enhancers at the endogenous location, the downstream enhancers were not blocked. Investigation of the 1A region revealed two clustered Su(Hw)-binding sites downstream of the yellow gene, named 1A-2, that bind Su(Hw) in vivo and possess enhancer blocking function. We propose that interaction between 1A-2 and the gypsy insulator permits activation of yellow expression by enhancers in the neighboring achaete-scute complex, causing an apparent absence of the block of the downstream yellow enhancers. Based on these data, we suggest that 1A-2 is an endogenous Su(Hw) insulator that separates regulatory domains within the Drosophila genome.
Animals↗
Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro.
The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhanced the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain was sufficient to form a higher-order complex with AML1/Ets-1, but exhibited only partial activity in transcription. We conclude that the T cell-enriched proteins assemble on the enhancer independently of CREB and function synergistically with CREB to activate the TCR alpha enhancer in a chromatin environment.
Base Sequence↗
Expression profiling and comparative genomics identify a conserved regulatory region controlling midline expression in the zebrafish embryo.
Differential gene transcription is a fundamental regulatory mechanism of biological systems during development, body homeostasis, and disease. Comparative genomics is believed to be a rapid means for the identification of regulatory sequences in genomes. We tested this approach to identify regulatory sequences that control expression in the midline of the zebrafish embryo. We first isolated a set of genes that are coexpressed in the midline of the zebrafish embryo during somitogenesis stages by gene array analysis and subsequent rescreens by in situ hybridization. We subjected 45 of these genes to Compare and DotPlot analysis to detect conserved sequences in noncoding regions of orthologous loci in the zebrafish and Takifugu genomes. The regions of homology that were scored in nonconserved regions were inserted into expression vectors and tested for their regulatory activity by transient transgenesis in the zebrafish embryo. We identified one conserved region from the connective tissue growth factor gene (ctgf), which was able to drive expression in the midline of the embryo. This region shares sequence similarity with other floor plate/notochord-specific regulatory regions. Our results demonstrate that an unbiased comparative approach is a relevant method for the identification of tissue-specific cis-regulatory sequences in the zebrafish embryo.
Animals↗
High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors.
Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.
Animals↗
Development of artificial chimerical gene regulatory elements specific for cancer gene therapy.
To achieve satisfactory outcome by the expression of therapeutic genes, it is of great importance to obtain efficient and high level of gene expression as well as minimizing inappropriate gene expression in non-target cells. To accomplish this goal for cancer gene therapy, we have evaluated the potential of cancer specific gene expression of functional promoter/enhancer elements in six putative cancer-specific genes (Tcf1alpha, C-Ha-Ras, CyclinE, Cdc25A, HK II, and hTert) using a luciferase reporter assay. Most of the reporter constructs showed higher activity in HepG2 cells than in non-transformed or stem cells, and, in particular, the hTert (E) or Tcf1alpha (T) regulatory element showed significantly higher activity. We have also constructed a series of artificial chimerical regulatory elements by combinatorial linking of E promoter and T enhancer. A dramatic decrease of activity was observed as the copy number of concatenated T/E regulatory elements increased. In contrast, in chimerical constructs containing two or three copies of regulatory elements of T/E, cell type preferential expression profiles were changed. Thus, both pGL3-TE and -TEE showed higher activity specifically in MCF7 breast cancer cells, whereas pGL3-TET showed moderate activity in several cancer cell lines of different origins. Our results demonstrate that although the transcriptional activities of synthetic promoters are weak, some cancer-specific regulatory elements are useful in developing optimized and systemic cancer-specific regulatory regions with potential application in targeted cancer cell therapy.
Amino Acid Motifs↗
Expression of the D-MEF2 transcription in the Drosophila brain suggests a role in neuronal cell differentiation.
D-MEF2 is a MADS domain transcription factor expressed in the cardiac, somatic, and visceral muscle cell lineages in the Drosophila embryo. Genetic studies have demonstrated that D-mef2 gene function is required for the proper differentiation of all three of these muscle types. We show that D-MEF2 is also expressed in a limited number of other cells types during development, including Kenyon cells present in the mushroom bodies of the Drosophila brain. This finding suggests a role for D-mef2 in neuron differentiation. To investigate D-mef2 expression in muscle and Kenyon cells, we assayed 26 kb of D-mef2 5'-flanking and intragenic DNA for regulatory sequences controlling the expression of the gene. Our results show that separable enhancer sequences direct D-mef2 gene expression in the myogenic and neuronal cell lineages. The identification of these regulatory DNAs provides a starting point for the analysis of transcriptional regulators controlling the cell-specific expression of D-mef2 and a means to address the function of D-mef2 in Kenyon cell differentiation.
Animals↗
An eye imaginal disc-specific transcriptional enhancer in the long terminal repeat of the tom retrotransposon is responsible for eye morphology mutations of Drosophila ananassae.
Optic morphology (Om) mutations of Drosophila ananassae are semidominant, neomorphic and nonpleiotropic, map to at least 22 loci scattered throughout the genome, and are associated with the insertion of the tom retrotransposon. Molecular and genetic analyses have revealed that eye morphology defects of Om mutants are caused by the ectopic or excessive expression of Om genes in the eye imaginal discs of third instar larvae. It is therefore assumed that the tom element carries tissue-specific gene regulatory sequences which enhance expression of the Om genes. In the present study, we examined whether or not the long terminal repeats (LTR) of the tom element contain such an eye imaginal disc-specific enhancer, using D. melanogaster transformants containing a lacZ gene ligated to the tom LTR. Analyses of lacZ gene expression in the eye imaginal discs of third instar larvae of 18 independently established transformant lines showed that the tom LTR was capable of enhancing lacZ expression in all the transformant lines, but the degree of enhancement varied between lines. In addition, the effect of the tom LTR lacZ gene evidently changed when the tom LTR construct was relocated to different chromosomal positions. On the basis of these findings, it is hypothesized that ectopic and excessive expression of the Om genes in the eye imaginal discs is induced by an eye imaginal disc-specific enhancer present in the tom LTR, the effect of which may be subject to chromosomal position effects.
Animals↗
SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution.
CRISPR mutagenesis screens conducted with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants but at a limited resolution due to the absence of protospacer adjacent motif (PAM) sequences. Here, leveraging the broad targeting scope of the near-PAMless SpRY variant, we have demonstrated that saturated SpRY mutagenesis and base editing screens can faithfully identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. We further extended this methodology to investigate a genome-wide association study (GWAS) locus at 10q22.1 associated with a red blood cell trait, where we identified potential enhancers regulating HK1 gene expression, despite not all of these enhancers exhibiting typical chromatin signatures. More importantly, our saturated base editing screens pinpoint multiple causal variants within this locus that would otherwise be missed by Bayesian statistical fine-mapping. Our approach is generally applicable to functional interrogation of all non-coding genomic elements while complementing other high-coverage CRISPR screens.
Humans↗
Listening to silence and understanding nonsense: exonic mutations that affect splicing.
Point mutations in the coding regions of genes are commonly assumed to exert their effects by altering single amino acids in the encoded proteins. However, there is increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing. Nonsense, missense and even translationally silent mutations can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency. As the splicing mechanisms that depend on exonic signals are elucidated, new therapeutic approaches to treating certain genetic diseases can begin to be explored.
Codon, Nonsense↗
Promoter analysis of RPE65, the gene encoding a 61-kDa retinal pigment epithelium-specific protein.
PURPOSE: To identify the functional promoter region and cis-acting elements that regulate the expression of RPE65, the retinal pigment epithelium (RPE)-specific gene responsible for certain forms of autosomal recessive childhood-onset severe retinal dystrophy. METHODS: A human genomic DNA clone containing the 5'-flanking region of RPE65 was isolated and, 4.0 kb proximal to the transcription start site, was sequenced and analyzed for the presence of transcription factor-binding sites. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the upstream sequence in the human RPE cell lines ARPE19 and D407, as well as in the SK-Mel-28 and HeLa cell lines. Specific DNA protein-binding sites present in the 340 bp upstream of the transcription start site were identified by DNase I footprint analysis. RESULTS: Sequence analysis places the polymorphic marker, D1S2803, within the RPE65 upstream region and identifies a number of sequences homologous to the gene encoding the cellular retinaldehyde-binding protein. Functional analysis indicates that basal promoter activity is conferred by the sequence from -83 to +39 and is approximately equivalent in all cell lines tested, with no other control elements detected in 3.6 kb of the upstream sequence. At least eight protected regions are identified in DNase I footprint assays, including sequences corresponding to the predicted TATA box, AP-4, and nuclear factor-1 DNA protein-binding sites. CONCLUSIONS: These findings localize the basal promoter activity of RPE65, identify potential cis-acting elements that act as positive regulators of gene expression, and suggest that additional regulatory elements are likely to be involved in restricting gene expression to the retinal pigment epithelium. Identification of promoter elements and genetic markers in the upstream sequence will enable the screening of patients with retinal degeneration for possible mutations that affect RPE65 expression.
Base Sequence↗
Gene and enhancer traps for gene discovery.
Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as beta-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.
Base Sequence↗