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Results for “Enhancer Elements, Genetic”

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At least 289 records · Page 16Linked to original sources

PubMed2003

In vivo regulation of the chicken cardiac troponin T gene promoter in zebrafish embryos.

The chicken cardiac troponin T (cTnT) gene is representative of numerous cardiac and skeletal muscle-specific genes that contain muscle-CAT (MCAT) elements within their promoters. We examined the regulation of the chicken cTnT gene in vivo in zebrafish embryos, and in vitro in cardiomyocyte, myoblast, and fibroblast cultures. Defined regions of the cTnT promoter were linked to the green fluorescent protein (GFP) gene for in vivo analysis, and the luciferase gene for in vitro analysis. Injection of the cTnT promoter constructs into fertilized zebrafish eggs resulted in GFP expression in both heart and skeletal muscle cells reproducing the pattern of expression of the endogenous cTnT gene in the chicken embryo. Promoter deletion analysis revealed that the cis-regulatory regions responsible for cardiac and skeletal muscle-specific expression functioned in an equivalent manner in both in vitro and in vivo environments. In addition, we show that mutation of the poly-ADP ribose polymerase-I (PARP-I) binding site adjacent to the distal MCAT element in the chicken cTnT promoter produced a non-cell-specific promoter in vitro and in the zebrafish. Thus, the PARP-I transcriptional regulatory mechanism that governs muscle specificity of the chicken cTnT promoter is conserved across several chordate classes spanning at least 350 million years of evolution.

Animals↗

PubMed2003

Splicing error in E1alpha pyruvate dehydrogenase mRNA caused by novel intronic mutation responsible for lactic acidosis and mental retardation.

An intronic point mutation was identified in the E1alpha PDH gene from a boy with delayed development and lactic acidosis, an X-linked disorder associated with a partial defect in pyruvate dehydrogenase (PDH) activity. Protein analysis demonstrated a corresponding decrease in immunoreactivity of the alpha and beta subunits of the PDH complex. In addition to the normal spliced mRNA product of the E1alpha PDH gene, patient samples contained significant levels of an aberrantly spliced mRNA with the first 45 nucleotides of intron 7 inserted in-frame between exons 7 and 8. The genomic DNA analysis found no mutation in the coding regions but revealed a hemizygous intronic G to A substitution 26 nucleotides downstream from the normal exon 7 5'-splice site. Splicing experiments in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which involves a switch from the use of the normal 5'-splice site (intron 7 position 1) to the cryptic 5'-splice site downstream of the mutation (intron 7 position 45). The intronic mutation is unusual in that it generates a consensus binding motif for the splicing factor, SC35, which normally binds to exonic enhancer elements resulting in increased exon inclusion. Thus, the aberrant splicing phenotype is most likely explained by the generation of a de novo splicing enhancer motif, which activates the downstream cryptic 5'-splice site. The mutation documented here is a novel case of intron retention responsible for a human genetic disease.

Acidosis, Lactic↗

PubMed2004

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development.

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.

Animals↗

PubMed2006

Transcriptional activation by extradenticle in the Drosophila visceral mesoderm.

decapentaplegic (dpp) is a direct target of Ultrabithorax (Ubx) in parasegment 7 (PS7) of the embryonic visceral mesoderm. We demonstrate that extradenticle (exd) and homothorax (hth) are also required for dpp expression in this location, as well as in PS3, at the site of the developing gastric caecae. A 420 bp element from dpp contains EXD binding sites necessary for expressing a reporter gene in both these locations. Using a specificity swap, we demonstrate that EXD directly activates this element in vivo. Activation does not require Ubx, demonstrating that EXD can activate transcription independently of homeotic proteins. Restoration is restricted to the domains of endogenous dpp expression, despite ubiquitous expression of altered specificity EXD. We demonstrate that nuclear EXD is more extensively phosphorylated than the cytoplasmic form, suggesting that EXD is a target of signal transduction by protein kinases.

Amino Acid Sequence↗

PubMed2004

Opposing inputs by Hedgehog and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc.

The sensory organs of the Drosophila adult leg provide a simple model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of longitudinal rows, a pattern that depends on periodic expression of the hairy gene (h) and the proneural genes achaete (ac) and scute (sc). Expression of ac in longitudinal stripes in prepupal leg discs defines the positions of the mechanosensory bristle rows. The ac/sc expression domains are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, we have analyzed a Hedgehog (Hh)- and Decapentaplegic (Dpp)-responsive enhancer of the h gene, which directs expression of h in a narrow stripe in the dorsal leg imaginal disc (the D-h stripe). Our studies suggest that the domain of D-h expression is defined by the overlap of Hh and high-level Dpp signaling. We find that the D-h enhancer consists of a Hh-responsive activation element (HHRE) and a repression element (REPE), which responds to the transcriptional repressor Brinker (Brk). The HHRE directs expression of h in a broad stripe along the anteroposterior (AP) compartment boundary. HHRE-directed expression is refined along the AP and dorsoventral axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp signaling is required to block Brk-mediated repression. This study elucidates a molecular mechanism for integration of the Hh and Dpp signals, and identifies a novel function for Brk as a repressor of Hh-target genes.

Animals↗

PubMed2005

bHLH-dependent and -independent modes of Ath5 gene regulation during retinal development.

In a wide range of vertebrate species, the bHLH transcription factor Ath5 is tightly associated with both the initiation of neurogenesis in the retina and the genesis of retinal ganglion cells. Here, we describe at least two modes of regulating the expression of Ath5 during retinal development. We have found that a proximal cis-regulatory region of the Xenopus Ath5 gene (Xath5) is highly conserved across vertebrate species and is sufficient to drive retinal-specific reporter gene expression in transgenic Xenopus embryos. Xath5 proximal transgene expression depended upon two highly conserved bHLH factor binding sites (E-boxes) as well as bHLH factor activity in vivo. However, we found that bHLH activity was not required for expression of a longer Xath5 transgene, suggesting that additional mechanisms contribute to Xath5 expression in vivo. Consistent with this, we showed that a more distal fragment that does not include the conserved proximal region is sufficient to promote transgene expression in the developing retina. In mouse, we found that a longer fragment of the cis-regulatory region of either the mouse or Xenopus Ath5 gene was necessary for transgene expression, and that expression of a mouse Math5 (Atoh7) transgene was not dependent upon autoregulation. Thus, despite extensive conservation in the proximal region, the importance of these elements may be species dependent.

Amino Acid Sequence↗

PubMed2001

Innervation of the ring gland of Drosophila melanogaster.

In insects, peptidergic neurons of the central nervous system regulate the synthesis of the main developmental hormones. Neuropeptides involved in this neuroendocrine cascade have been identified in lepidopterans and dictyopterans. Since these organisms are not suitable for genetic research, we identified peptidergic brain neurons innervating the ring gland in Drosophila melanogaster. In larvae of Drosophila, ecdysteroids and juvenile hormones are produced by the ring gland, which is composed of the prothoracic gland, the corpus allatum, and the corpora cardiaca. Using the GAL4 enhancer trap system, we mapped those neurons of the central nervous system that innervate the ring gland. Eleven groups of neurosecretory neurons and their target tissues were identified. Five neurons of the lateral protocerebrum directly innervate the prothoracic gland or corpus allatum cells of the ring gland and are believed to regulate ecdysteroid and juvenile hormone titers. Axons of the circadian pacemaker neurons project onto dendritic fields of these five neurons. This connection might be the neuronal substrate of the circadian rhythms of molting and metamorphosis in Drosophila. Most of the neurons presented here have not been described before. The enhancer trap lines labeling them will be valuable tools for the analysis of neuronal as well as genetic regulation in insect development.

Animals↗

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