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PubMed1991

Mutations within the Ddc promoter alter its neuron-specific pattern of expression.

The Drosophila dopa decarboxylase gene (Ddc) is expressed in a reproducible set of approximately 150 neurons, and in a subset of the glia of the third instar larva's central nervous system (CNS). Expression in this pattern requires a cell type-specific neuronal enhancer/glial repressor region located 1000 bp from the transcriptional start site, and specific sequences within the promoter. We have used mutagenesis in vitro and P-element-mediated transformation to examine the role of the promoter, particularly its major CNS activator sequence (element I), in the generation of the wildtype expression pattern. Immunohistological analysis of these transgenic strains demonstrates that particular deletion mutations shift the site of transgene expression to a set of neurons which do not express Ddc at detectable levels in wild-type larvae. Transgene expression in these strains may be driven by a previously undetected activator sequence. Our data also suggest that glial expression may be driven by the same activator sequences that drive expression in the hypoderm.

Animals↗
PubMed2000

GATA2 is required for the generation of V2 interneurons.

During embryogenesis, transcription factor GATA2 is expressed in a variety of distinct cell types, and earlier experiments showed that GATA2 is a vital regulator of both hematopoiesis and urogenital development. Despite the fact that GATA2 is expressed early and abundantly in the nervous system, there has been no demonstration of its direct participation in neurogenesis. We show here that GATA2 is expressed in the ventral spinal cord exclusively in newly generated V2 interneurons, suggesting that GATA2 might be required for the generation of this discrete neuronal population. Proof for this hypothesis was provided by showing that the number of cells expressing V2 neuronal markers was drastically diminished in gata2 null mutant embryos. The tissue-specific enhancer that directs gata2 transcription specifically in V2 neurons was localized to a 190 bp intragenic element lying within gata2 intron 5, and this element is both necessary and sufficient to confer GATA2 spinal cord expression. The identification of a V2-specific enhancer should allow fundamental new insight into the genetic hierarchy of regulatory events that govern neurogenesis in a well-defined cell lineage.

Animals↗
PubMed1992

Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix.

The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins. Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.

Alkylation↗
PubMed1995

Transcriptional silencing by the Polycomb protein in Drosophila embryos.

Polycomb group (Pc-G) proteins act to keep homeotic genes stably and heritably silenced during Drosophila development. Here, it is shown that Polycomb (Pc), one of the Pc-G proteins, acts as a transcriptional silencer in Drosophila embryos if tethered to reporter genes by the DNA binding domain of GAL4 (i.e. as a GAL-Pc fusion protein). The results suggest that silencing by GAL-Pc requires the C-terminal portion of Pc, but not the chromodomain. If a pulse of Gal-Pc is provided, synthetic reporter genes are repressed, though only transiently. In contrast, reporter genes containing homeotic gene sequences remain stably and heritably silenced in a Pc-G gene-dependent fashion, even when GAL-Pc is no longer present. This implies that GAL-Pc recruits Pc-G proteins to DNA and suggests that maintenance of silencing requires the anchoring of Pc-G proteins to specific cis-regulatory sequences present in homeotic genes. The extent of DNA over which the Pc-G machinery acts is quite selective, as silencing established on one enhancer does not necessarily 'spread' to a juxtaposed synthetic enhancer.

ATP-Binding Cassette Transporters↗
PubMed1995

Tissue-specific activation of tumor marker glutathione transferase P transgenes in transgenic rats.

By means of transgenic rats, we have recently shown that the GPEI enhancer of the glutathione transferase P (GST-P) gene, which has two one-base-missmatched AP-1 sites locating palindromically with three-base spacing in between, is sufficient for conferring tumor-specific activation of the gene in vivo. It is noted that there is another consensus AP-1 site near the promoter of this gene. By using seven independent transgenic rats, bearing distinct areas of the GST-P gene that are connected to the chloramphenicol acetyltransferase (CAT) coding sequence, we analyzed CAT expression in various tissues (brain, lung, liver, kidney, spleen) in these transgenic rats. We found that the ECAT gene, which has sufficient of the upstream regulatory region (approx. 2.9 kb) of the gene containing GPEI, is trans-activated in the kidney and lung of transgenic rats in a similar manner to endogenous GST-P. When either the GPEI core sequence or the AP-1 site near the promoter is deleted, CAT expression decreases to almost background level. Substitution of the GPEI core or the AP-1 site near the promoter to this silent construct (5CATGPEIcore) reconstituted CAT expression in the transgenic rats. In these rats, CAT was expressed in the brain and lung rather than in the kidney, showing a somewhat different pattern from the endogenous GST-P. In the brain tissue of the 5CATGPEIcore transgenic rat, CAT was demonstrated in the glia cells, which is consistent with endogenous GST-P expression. These results suggest that a relatively long upstream region (approx. 2.9 kb) is required for tissue-specific expression of the GST-P gene and that GST-P expression in the brain may be regulated differently from its expression in other organs.

Animals↗
PubMed2003

Analysis of the biological functions of a doublesex homologue in Bombyx mori.

We have previously reported that Bmdsx, a homologue of the sex-determining gene doublesex ( dsx), was sex-specifically expressed in various tissues of the silkworm. The primary transcript of Bmdsx is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, we expressed BmDSXF in males from a ubiquitous promoter and examined its regulatory activities. We show that BmDSXF functions as a positive regulator of the hexameric storage protein termed SP1 and vitellogenin genes that are predominantly expressed in females. We also show that expression of Bmdsx(F) in males results in the repression of the pheromone-binding protein gene that is preferentially expressed in males. Gel-mobility shift assays demonstrated that BmDSX proteins bind to the sequence (ACATTGT) between -95 and -89 nt relative to the transcriptional initiation site of the vitellogenin gene. These results strongly suggest that Bmdsx is a final regulatory gene in the hierarchy of regulatory genes controlling the expression of female-specific protein in Bombyx mori.

Animals↗
PubMed1994

Direct regulation of decapentaplegic by Ultrabithorax and its role in Drosophila midgut morphogenesis.

Drosophila homeotic genes encode transcription factors thought to control segmental identity by regulating expression of largely unknown target genes. The formation of the second midgut constriction requires the Ultrabithorax (Ubx) and abdominal-A (abd-A) homeotic genes and decapentaplegic (dpp), a gene encoding a member of the TGF beta family of proteins. We identified a 674 bp enhancer of dpp controlling its expression in the second constriction domain of the visceral mesoderm (parasegment 7). Normal enhancer function requires positive regulation by Ubx and negative regulation by abd-A. This enhancer contains UBX- and ABD-A-binding sites defined in vitro. By generating complementary alterations of the binding sites and the binding specificity of UBX, we show that Ubx directly regulates dpp expression. These regulatory interactions are relevant to normal development, because a transgene made with this enhancer driving a dpp transcription unit rescues the second midgut constriction and larval lethality phenotypes of dpps mutations.

Animals↗
PubMed2003

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction.

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene.

Animals↗
PubMed2002

Enhancer action in trans is permitted throughout the Drosophila genome.

Interactions between paired homologous genes can lead to changes in gene expression. Such trans-regulatory effects exemplify transvection and are displayed by many genes in Drosophila, in which homologous chromosomes are paired somatically. Transvection involving the yellow cuticle pigmentation gene can occur by at least two mechanisms, one involving the trans-action of enhancers on a paired promoter and a second involving pairing-mediated bypass of a chromatin insulator. A system was developed to evaluate whether the action of the yellow enhancers in trans could be reconstituted outside of the natural near telomeric location of the yellow gene. To this end, transgenic flies were generated that carried a yellow gene modified by the inclusion of strategically placed recognition sites for the Cre and FLP recombinases. Independent action of the recombinases produced a pair of derivative alleles, one enhancerless and the other promoterless, at each transgene location. Transvection between the derivatives was assessed by the degree of interallelic complementation. Complementation was observed at all eight sites tested. These studies demonstrate that yellow transvection can occur at multiple genomic locations and indicate that the Drosophila genome generally is permissive to enhancer action in trans.

Alleles↗
PubMed1996

An enhancer LEF-1/TCF-1 site is essential for insertion site-independent transgene expression in thymus.

Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein-protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.

Aminohydrolases↗
PubMed2001

Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family.

The activity of the DAF-2 insulin-like receptor is required for Caenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weak daf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of the ins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family.

Amino Acid Sequence↗
PubMed2006

A genetic screen in Drosophila for genes interacting with senseless during neuronal development identifies the importin moleskin.

Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens.

Animals↗
PubMed2003

Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.

Little has been understood about the underlying mechanisms that generate the morphological diversity of dendritic trees. Dendritic arborization neurons in Drosophila provide an excellent model system to tackle this question, and they are classified into classes I-IV in order of increasing arbor complexity. Here we have developed transgenic green fluorescent protein markers for class I or class IV cells, which allowed time-lapse recordings of dendritic birth in the embryo, its maturation processes in the larva, and lesion-induced reactions. The two classes used distinct strategies of dendritic emergence from the cell body and branching, which contributed to differences in their basic arbor patterns. In contrast to the class I cells examined, one cell of class IV, which was a focus in this study, continued to elaborate branches throughout larval stages, and it was much more capable of responding to the severing of branches. We also investigated the cellular basis of field formation between adjacent class IV cells. Our results support the fact that class-specific inhibitory interaction is necessary and sufficient for tiling and confirmed that this intercellular communication was at work at individual dendrodendritic interfaces. Finally, this inhibitory signaling appeared to play a central role when arbors of adjacent cells started meeting midway between the cells and until the body wall became partitioned into abutting, minimal-overlapping territories.

Animals↗
PubMed1997

Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene.

Regulated expression of recombinant genes in CD4+ cells is an important objective for gene therapy of AIDS, as these cells represent the principal target for viral replication of human immunodeficiency virus (HIV). We report here that specific combinations of CD4 cell-specific and viral regulatory elements can enhance expression of an antiviral gene product. Different viral regulatory elements were incorporated into a previously reported CD4 locus control region to increase the expression of reporter genes in T and monocytic cell lines. The CD4-specific regulatory elements were included to enhance expression in CD4 cells, and viral regulatory regions, including the cytomegalovirus immediate-early (CMV IE) upstream enhancer, which contains the kappa B and Ap1 regulatory elements and a Tat-responsive element of the HIV type 1 long terminal repeat, were used to increase gene expression and modulate its activity in response to viral infection. In transient transfection assays, this vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone. Expression of an inhibitory form of the Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4+ peripheral blood mononuclear cells. The combination of CD4 lineage-specific and viral regulatory elements will facilitate the development of more effective antiviral genetic strategies for AIDS.

CD4 Antigens↗
PubMed1999

A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner.

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

Animals↗
PubMed1995

An enhancer deletion affects both H19 and Igf2 expression.

The distal end of mouse Chromosome 7 contains four tightly linked genes whose expression is dependent on their parental inheritance. Mash-2 and H19 are expressed exclusively from the maternal chromosome, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf2) are paternally expressed. The identical expression during development of the 3'-most genes in the cluster, Igf2 and H19, led to the proposal that their imprinting was mechanistically linked through a common set of transcriptional regulatory elements. To test this hypothesis, a targeted deletion of two endoderm-specific enhancers that lie 3' of H19 was generated by homologous recombination in embryonic stem cells. Inheritance of the enhancer deletion through the maternal lineage led to a loss of H19 gene expression in cells of endodermal origin, including cells in the liver, gut, kidney, and lung. Paternal inheritance led to a very similar loss in the expression of Igf2 RNA in the same tissues. These results establish that H19 and Igf2 utilize the same endoderm enhancers, but on different parental chromosomes. Mice inheriting the enhancer deletion from fathers were 80% of normal size, reflecting a partial loss-of-function of Igf2. The reduction was uniformly observed in a number of internal organs, indicating that insulin-like growth factor II (IGFII), the product of Igf2, acts systemically in mice to affect prenatal growth. A modest decline in Ins-2 RNA was observed in the yolk sac. In contrast Mash-2, which is expressed in spongiotrophoblast cells of the placenta, was unaffected by the enhancer deletion.

Animals↗