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Results for “Enhancer Elements, Genetic”

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PubMed1998

Drosophila homeobox gene eve enhances trol, an activator of neuroblast proliferation in the larval CNS.

The regulation of stem cell division by developmental cues is critical for the assembly and function of multicellular organisms. Stem cell division in the Drosophila brain is controlled by trol, which is required for activation of proliferation by quiescent neuroblasts at the appropriate stage of larval development. We show that the transcriptional regulator eve is part of the trol activation pathway by identifying eve as a dominant enhancer of a weak trol allele, trolb22. Known eve mutations are capable of enhancing the lethality of trolb22 and uncovering a defective neuroblast proliferation phenotype. Additionally, genetic and molecular analysis reveals that an independent mutation which acts as a dominant enhancer of trol is also an allele of eve. The enhancement of trolb22 lethality can be suppressed by the presence of an eve transgene. Interestingly, extra copies of eve supplied by the eve transgene also enhance trolb22 lethality, suggesting that the level of Eve protein may be critical for neuroblast activation. Finally, activation of neuroblast proliferation is normal in eve4 heterozygotes, suggesting that the proliferation defect observed in trolb22;eve/+ animals is due to a synergistic interaction.

Alleles↗
PubMed1996

Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo.

Previous studies on the regulation of the segmentation gene even-skipped (eve) have centered on the transcription of stripe 2. Here, we characterize another enhancer module contained within the complex eve promoter that directs expression of stripes 3 and 7. This enhancer is approximately 500 bp in length and maps approximately 3.3 kb upstream of the transcription start site. The stripe 3 + 7 enhancer appears to be regulated by one or more ubiquitously distributed activators, including components of a JAK-Stat pathway. The two-stripe pattern results via multiple tiers of repressors which delimit this ubiquitous activation. The zinc finger repressor hunchback appears to be responsible for establishing the anterior border of stripe 3 and the posterior border of stripe 7. knirps, a member of the nuclear receptor family of transcription factors, appears to establish the posterior border of stripe 3 and the anterior border of stripe 7. Activator and repressor proteins bind in vitro to several sites within the enhancer. These findings suggest a general model for the regulation of segmentation stripes, whereby enhancers integrate positional information provided by broadly distributed activators and spatially restricted repressors.

Alleles↗
PubMed2025

Redistribution of super-enhancers promotes malignancy in human hepatocellular carcinoma.

INTRODUCTION: Super-enhancers (SEs) are defined as the regulatory region where intensive transcriptional cofactors bind. Dysregulation of SEs is related to multiple diseases, however, its role in hepatocellular carcinoma (HCC) remains elusive. OBJECTIVES: This work aimed to reveal the dysregulation of SEs in HCC and the therapeutic potential for HCC treatment. METHODS: Fifteen HCC and twelve paracancerous samples underwent chromatin immunoprecipitation (ChIP) sequencing targeting H3K27ac, and subsequently the SEs were identified by the Rank Ordering of Super-Enhancers algorithm. Differential SEs featured by tumor or paracancerous tissues were identified, and cross-referenced with the differential expression genes and prognosis-related genes in 2 independent public or in-house HCC cohorts. The SE region of HSPA4 was deleted in the genome of HCCLM3 cell by CRISPR-Cas9, named HSPA4-SE-KO cells. The potential druggable transcriptional factors were identified by CRCmapper, GeneMANIA and Drug Gene Interaction Database (DGID). RESULTS: Five targets, including CDKN2C, HSPA4, GGH, PDGFA, and CAP2, were identified as HCC-gain SEs with oncogenic potential, which were further validated experimentally by SE inhibitors and ChIP targeting H3K27ac and BRD4. Cell proliferation and migration assays further confirmed that silencing of these HCC-gain SEs significantly suppressed the malignant phenotype of HCC cell lines. HSPA4 appeared strongest oncogenic functions among these targets, which was further verified by HCC mouse xenograft models and clinical sample investigation. Moreover, HSPA4-SE-KO cells obtained significantly suppressed HSPA4 expression and retarded tumorigenic capability. Finally, dysregulation of transcriptional factors engaged in the oncogenic role of SEs, and Danthron that targeting RXRA were identified from DGID for HCC treatment. CONCLUSION: The dysregulated SE landscape of HCC promoted the malignancy phenotype by the upregulation of oncogenes, and SE-regulatory network might be potential drug targets for HCC treatment. Our study deepened the insight of epigenetic dysregulation in HCC, offering the groundwork for SEs as potential therapeutic targets of HCC treatment.

Humans↗
PubMed2006

Temporal regulation of late expression of Bar homeobox genes during Drosophila leg development by Spineless, a homolog of the mammalian dioxin receptor.

The spatial and temporal regulation of genes encoding transcription factors is essential for the proper development of multicellular organisms. In Drosophila leg development, the distal-most tarsus (ta5) is specified by the strong expression of a pair of Bar homeobox genes in late third instar. This expression is regulated under the control of the ta5 enhancer activated by Bar. No activation of the ta5 enhancer, however, occurs in early third instar when considerable Bar is produced. The ta5 enhancer was comprised of a basal enhancer required for driving Bar expression and a negative regulatory motif serving as a binding site for the heterodimer of Spineless and Tango, homologs of the mammalian dioxin receptor and aryl hydrocarbon nuclear translocator, respectively. The spineless and tango were essential for suppressing the basal enhancer activation in early third instar. The spineless was transiently expressed in early third instar in the Bar expression domain. ta5 Bar expression may thus be temporally regulated through transient inhibition of premature activation of the basal enhancer via specific binding of the Spineless/Tango heterodimer to the negative regulatory motif in early third instar and subsequent release from the inhibition due to the disappearance of spineless expression at later stages.

Animals↗
PubMed2000

LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator.

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.

Animals↗
PubMed1996

Short-range transcriptional repressors mediate both quenching and direct repression within complex loci in Drosophila.

The early Drosophila embryo provides a unique system for the analysis of transcriptional repression since a broad spectrum of repressors are distributed in spatially distinct patterns. Krüppel (Kr) and snail (sna), two zinc finger repressors, are essential for segmentation and for the establishment of the mesoderm/neuroectoderm boundary, respectively. Both repressors were examined in the context of synthetic gene complexes containing modular promoters and divergently transcribed reporter genes. These studies indicate that Kr and sna function as short-range repressors, which can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. When located within an upstream enhancer, the repressor locally quenches nearby activators and permits other enhancers to interact with the transcription complex (enhancer autonomy). In contrast, when bound to promoter-proximal regions the repressor functions in a dominant fashion and blocks multiple enhancers. Local quenching and dominant repression require close linkage (<100 bp) of the repressor with either upstream activators or the transcription complex. These studies establish short-range repression as a flexible form of gene regulation and suggest that the key distinction among repressors is their range of action.

Animals↗
PubMed1993

[Genetic analysis of the interaction of mutations in two genes, controlling expression of MDG4].

Transcription of the retrovirus-like mobile element mdg4 (gypsy) in controlled by the protein product of the suppressor of Hairy wing which interacts with the mdg4 enhancer, and by the protein product of the mdg4 modifier which seems to interact with the su(Hw) protein. In the present work, we studied interaction of mod(mdg4) and su(Hw) mutations in phenotypic expression of mutations induced by mdg4 insertion. Different types of inhibitory effect of mdg4 enhancer on transcription of the target gene were detected. Most frequently, the inhibitory depends on the presence of the mod(mdg4) protein. On of the two acidic domains of su(Hw) protein may be removed without changing the effect. Additional removal of the leucine zipper domain strongly reduces the effect. The second type of inhibition depends of or decrease in the concentration of the mod(mdg4) protein. The su(Hw) protein should have both acidic domains in this case. Sometimes, the su(Hw) protein lacking the C-end acidic domain may activate transcription of a target gene, and this effect does not depend on the presence or absence of the mod(mdg4) protein. The effect may be increased by removal of leucine zipper. Binding of the mod(mdg4) protein to the su(Hw) protein does not depend of the C-end acidic domain and leucine zipper.

Animals↗
PubMed1998

Development of a condensed locus control region cassette and testing in retrovirus vectors for A gamma-globin.

Retrovirus vectors for A gamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various A gamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, A gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the beta pr.A gamma cassettes.

Animals↗
PubMed2005

Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites.

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.

Amino Acid Sequence↗
PubMed2000

Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases.

Exonic splicing enhancers (ESEs) are discrete sequences within exons that promote both constitutive and regulated splicing. The precise mechanism by which ESEs facilitate the assembly of splicing complexes has been controversial. However, recent studies have provided insights into this question and have led to a new model for ESE function. Other recent work has suggested that ESEs are comprised of diverse sequences and occur frequently within exons. Ominously, these latter studies predict that many human genetic diseases linked to mutations within exons might be caused by the inactivation of ESEs.

Antigens, Nuclear↗
PubMed2006

Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways.

Most heritable traits, including disease susceptibility, are affected by interactions between multiple genes. However, we understand little about how genes interact because very few possible genetic interactions have been explored experimentally. We have used RNA interference in Caenorhabditis elegans to systematically test approximately 65,000 pairs of genes for their ability to interact genetically. We identify approximately 350 genetic interactions between genes functioning in signaling pathways that are mutated in human diseases, including components of the EGF/Ras, Notch and Wnt pathways. Most notably, we identify a class of highly connected 'hub' genes: inactivation of these genes can enhance the phenotypic consequences of mutation of many different genes. These hub genes all encode chromatin regulators, and their activity as genetic hubs seems to be conserved across animals. We propose that these genes function as general buffers of genetic variation and that these hub genes may act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.

Animals↗
PubMed1999

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 microgram/ml of canine factor IX was detected in the plasma of mice injected with 5.6 x 10(11) particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.

Animals↗
PubMed1994

Modification of the Drosophila heterochromatic mutation brownDominant by linkage alterations.

The variegating mutation brownDominant (bwD) of Drosophila melanogaster is associated with an insertion of heterochromatin into chromosome arm 2R at 59E, the site of the bw gene. Mutagenesis produced 150 dominant suppressors of bwD variegation. These fall into two classes: unlinked suppressors, which also suppress other variegating mutations; and linked chromosome rearrangements, which suppress only bwD. Some rearrangements are broken at 59E, and so might directly interfere with variegation caused by the heterochromatic insertion at that site. However, most rearrangements are translocations broken proximal to bw within the 52D-57D region of 2R. Translocation breakpoints on the X chromosome are scattered throughout the X euchromatin, while those on chromosome 3 are confined to the tips. This suggests that a special property of the X chromosome suppresses bwD variegation, as does a distal autosomal location. Conversely, two enhancers of bwD are caused by translocations from the same part of 2R to proximal heterochromatin, bringing the bwD heterochromatic insertion close to the chromocenter with which it strongly associates. These results support the notion that heterochromatin formation at a genetic locus depends on its location within the nucleus.

Animals↗
PubMed2005

A new strategy for efficient in vivo screening of mutagenized Drosophila embryos.

The analysis of mutants is an indispensable approach towards characterizing gene function. Combining several tools of Drosophila genetics, we designed a new strategy for a mutagenesis screen which is fast, easy-to-apply, and cheap. The combination of a cell-specific Gal4 line with an upstream activating sequence-green fluorescent protein (UAS-GFP) allows the in vivo detection of the cells or tissues of interest without the need for fixation and staining. To further simplify and accelerate the screening procedure, we generated recombinant flies that carry the Gal80 transgene in balancer chromosomes. Gal80 inactivates Gal4; and thus prevents GFP-expression during embryonic and postembryonic development in all individuals carrying the balancer chromosomes. This allows for an easy distinction in vivo between heterozygous and homozygous mutants, the latter being the only ones expressing GFP. Since most of the fly strains and balancer chromosomes can be substituted, this method is suitable for nearly any mutagenesis screen that does not have major restrictions.

Animals↗
PubMed2000

Analysis of vertebrate SCL loci identifies conserved enhancers.

The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.

Amino Acid Sequence↗
PubMed1988

Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.

Animals↗
PubMed1996

Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila.

Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.

Animals↗