Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300.
(17/5432)
Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations. (+info)
Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation.
(18/5432)
While the transmembrane protein Notch plays an important role in various aspects of development, and diseases including tumors and neurological disorders, the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and Enhancer-of-split homologues) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, Hes1-null, Hes5-null and Hes1-Hes5 double-null mouse embryos. We found that caNotch, which induced the endogenous Hes1 and Hes5 expression, inhibited neuronal differentiation in the wild-type, Hes1-null and Hes5-null background, but not in the Hes1-Hes5 double-null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in regulation of mammalian neuronal differentiation. (+info)
The su(Hw) insulator can disrupt enhancer-promoter interactions when located more than 20 kilobases away from the Drosophila achaete-scute complex.
(19/5432)
Here we report that the su(Hw) insulator may not necessarily separate promoters from enhancers to allow inhibition of transcription by the su(Hw) protein. For this purpose we used the strains of Drosophila melanogaster which carry inversion of the region containing the yellow gene and the achaete-scute complex (AS-C). Despite the reverse orientation of the region, the AS-C enhancers continue to activate achaete and scute gene expression. The su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong suppression of achaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers. (+info)
Ectopic expression of individual E(spl) genes has differential effects on different cell fate decisions and underscores the biphasic requirement for notch activity in wing margin establishment in Drosophila.
(20/5432)
A common consequence of Notch signalling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. We expressed each individual E(spl) gene using the GAL4-UAS system in order to analyse their effect in a number of cell fate decisions taking place in the wing imaginal disk. We focussed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way, namely they antagonize neural precursor and vein fates. Yet, the efficacy of this antagonism is quite distinct: E(spl)mbeta has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. We have discerned two phases within this process based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns. (+info)
Distinct 5' SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor binding sites.
(21/5432)
The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal cord. In addition we have demonstrated that GATA factor binding sites are essential for neural expression of the SCL constructs. The midbrain element was particularly powerful and axonal lacZ expression revealed the details of axonal projections, thus implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene was highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibited a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions. (+info)
Rib truncations and fusions in the Sp2H mouse reveal a role for Pax3 in specification of the ventro-lateral and posterior parts of the somite.
(22/5432)
The splotch (Pax3) mouse mutant serves as a model for developmental defects of several types, including defective migration of dermomyotomal cells to form the limb musculature. Here, we describe abnormalities of the ribs, neural arches, and acromion in Sp2H homozygous embryos, indicating a widespread dependence of lateral somite development on Pax3 function. Moreover, the intercostal and body wall muscles, derivatives of the ventrolateral myotome, are also abnormal in Sp2H homozygotes. Pax3 is expressed in the dermomyotome, but not in either the sclerotome or the myotome, raising the possibility that Pax3-dependent inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome and myotome. Support for this idea comes from analysis of gene expression markers of lateral sclerotome (tenascin-C and scleraxis) and myotome (myogenin, MyoD, and Myf5). All exhibit ventrally truncated domains of expression in Sp2H homozygotes, potentially accounting for the rib and intercostal muscle truncations. In contrast, the medial sclerotomal marker Pax1 is expressed normally in mutant embryos, arguing that Pax3 is not required for development of the medial sclerotome. Most of the somitic markers show ectopic expression in anteroposterior and mediolateral dimensions, suggesting a loss of definition of somite boundaries in splotch and explaining the rib and muscle fusions. An exception is Myf5, which is not ectopically expressed in Sp2H homozygotes, consistent with the previous suggestion that Pax3 and Myf5 function in different pathways of skeletal myogenesis. PDGFalpha and its receptor are candidates for mediating signalling between myotome and sclerotome. We find that both genes are misexpressed in Sp2H embryos, suggesting that PDGFalpha/PDGFRalpha may function downstream of Pax3, accounting for the close similarities between the splotch and Patch mutant phenotypes. Our findings point to additional regulatory functions for the Pax3 transcription factor, apart from those already demonstrated for development of the neural tube, neural crest, and dermomyotome. (+info)
Expression of the basic helix-loop-helix protein REBalpha in rat testicular Sertoli cells.
(23/5432)
Sertoli cell differentiation is initiated in the embryo to promote testicular development and male sex determination. In the adult, Sertoli cells are critical for maintenance of the spermatogenic process. Previously, Sertoli cell differentiation has been shown to be regulated in part by basic helix-loop-helix (bHLH) transcription factors. This was based on the observation that promoters of a number of Sertoli cell genes contained bHLH-responsive E-box response elements and that overexpression of Id, a negatively acting HLH protein, down-regulates Sertoli cell differentiated functions. Analysis of Sertoli cell bHLH proteins demonstrated the expression of REBalpha in Sertoli cells. REBalpha and REBbeta are spliced variants of the REB gene that is implicated in cell-specific gene expression as part of dimeric bHLH complexes acting on E-box response elements. Although both the transcripts of the REB gene are widely expressed, differential expression of the REB gene transcripts REBalpha and REBbeta has been shown. In the current study, a polymerase chain reaction (PCR)-based approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REBalpha is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REBalpha or REBbeta demonstrated that Sertoli cells express only REBalpha, not REBbeta. REBbeta was present in the RNA samples obtained from whole testis, suggesting expression in other testicular cell types. A Northern blot analysis of RNA from Sertoli cells treated with or without FSH or cAMP demonstrated that REBalpha is not hormone responsive. REBalpha was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REBalpha is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REBalpha in Sertoli cells was demonstrated with an E-box gel shift with Sertoli cell nuclear extracts. The E-box gel shift was found to contain REBalpha and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REBalpha isoform of the REB gene. The results are discussed in relation to the possibility that Sertoli cells may express a cell-specific bHLH protein that can preferentially dimerize with REBalpha. (+info)
Photomophogenesis: Phytochrome takes a partner!
(24/5432)
How light signals are transduced by phytochromes is still poorly understood. Recent studies have provided evidence that a PAS domain protein, PIF3, physically interacts with phytochromes, plays a role in phytochrome signal transduction and might be a component of a novel signalling pathway in plants. (+info)