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Results for “regulatory module”

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PubMed1999

Mechanism of intrinsic transcription termination and antitermination.

Gene expression is modulated by regulatory elements that influence transcription elongation by RNA polymerase: terminators that disrupt the elongation complex and release RNA, and regulators that overcome termination signals. RNA release from Escherichia coli RNA polymerase can be induced by a complementary oligonucleotide that replaces the upstream half of the RNA hairpin stem of intrinsic terminator transcripts, implying that RNA hairpins act by extracting RNA from the transcription complex. A transcription antiterminator inhibits this activity of oligonucleotides and therefore protects the elongation complex from destabilizing attacks on the emerging transcript. These effects illuminate the structure of the complex and the mechanism of transcription termination.

Base Pairing↗

PubMed1992

Simian virus 40 T antigen activates the late promoter by modulating the activity of negative regulatory elements.

Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.

Animals↗

PubMed1996

A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression is dependent on the transactivator protein Tat and an RNA element extending from the transcription initiation site to +57 known as TAR. TAR forms a stable RNA secondary structure which is critical for high levels of HIV-1 gene expression and efficient viral replication. Using a genetic approach, we isolated HIV-1 mutants in TAR that were competent for high levels of gene expression but yet were markedly defective for viral replication. Single-cycle infections with these viruses demonstrated that they were defective in the initiation of reverse transcription. Additional mutational analysis revealed a variety of other HIV-1 TAR mutants with the same defective phenotype. Thus, in addition to the well-characterized role of the primer binding site, other RNA elements within the HIV-1 genome are also critical in the regulation of reverse transcription. These studies demonstrate that HIV-1 TAR RNA is a key regulator of the reverse transcription and illustrate how a unique RNA structure can modulate diverse regulatory processes in the HIV-1 life cycle crucial for efficient viral replication.

Base Sequence↗

PubMed2006

Active chromatin hub of the mouse alpha-globin locus forms in a transcription factory of clustered housekeeping genes.

RNA polymerases can be shared by a particular group of genes in a transcription "factory" in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse alpha-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active alpha1 and alpha2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse alpha-globin cluster. In fetal liver cells, the active alpha1 and alpha2 genes, but not the inactive zeta gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse alpha-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active alpha1 and alpha2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the zeta gene promoter, which is specifically repressed during development, is much lower than that at the alpha1 and alpha2 promoters. Thus, the mouse alpha-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate alpha-globin gene expression.

Animals↗

PubMed1994

Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae.

The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription. To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase. Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type. Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3. Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region. Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation. Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies. MSN3 is the same gene as STD1, a multicopy suppressor of defects caused by overexpression of the C terminus of TATA-binding protein (R. W. Ganster, W. Shen, and M. C. Schmidt, Mol. Cell. Biol. 13:3650-3659, 1993). Taken together, these data suggest that MSN3 modulates the regulatory response to glucose and may couple the SNF1 pathway to transcription.

Adaptor Proteins, Signal Transducing↗

PubMed1991

RNA transcription and translation in the hearts of normal and cardiomyopathic Syrian hamsters.

The cardiomyopathic Syrian hamster has an autosomal recessive defect that results in the development of an early onset cardiac myopathy leading to cardiac dysfunction and, eventually, complete heart failure. To assess the regulatory mechanisms modulating gene expression in the normal and myopathic myocardium, we investigated both RNA transcription and translation. Our results indicated that the incorporation of [3H]UMP into myocardial cell nuclear RNA decreased 10-fold from 7 to 210 days of age in the normal Syrian hamster. The incorporation of [3H]UMP was approximately 50% lower in the cardiomyopathic as compared with the normal Syrian hamster. RNA translation, as assessed by rabbit reticulocyte lysate in vitro translation, indicated that a coordinated 50% decrease in RNA translation occurred in normal Syrian hamster from 7 to 210 days of age. A further reduction of 20% in translation was found in cardiomyopathic Syrian hamster ventricular RNA translation as compared with matched random bred control groups. Two-dimensional polyacrylamide gel analysis of cell-free translated protein products demonstrated two myocardial peptides that were found to be consistently altered when the normal and cardiomyopathic Syrian hamsters were compared. These results indicate that transcription and translation decrease with age and that these processes are further downregulated, in an additive manner, with the genesis of the disease process.

Aging↗

PubMed1999

Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments.

The hypothesis that ovarian sex hormone deficiency affects cardiac myofilament activation was tested. Chemically skinned ventricular trabeculae and single soleus muscle fibers were prepared from 10- and 14-wk ovariectomized and control rats. Tension-pCa (-log [Ca(2+)]) relations of left ventricular trabeculae and soleus fibers were compared to test whether thin filament proteins are potential sites of modulated activation. Trabeculae from ovariectomized rats exhibited a significant increase in Ca(2+) sensitivity with no change in maximal tension-generating ability. In contrast, soleus fibers demonstrated no shift in Ca(2+) sensitivity but generated significantly less maximal tension. No changes in thin filament protein isoform expression or loss of thin filament proteins were apparent in the trabeculae or soleus fibers from ovariectomized rats. Although not directly tested, our results are consistent with a possible modulation of regulatory proteins (e.g., cardiac troponin I) to account for the observed change in myofilament responsiveness of hearts from ovariectomized rats. Other possible mechanisms for the altered myocardial Ca(2+) sensitivity after ovariectomy are discussed.

Actin Cytoskeleton↗

PubMed1996

Gastric endocrine cells: gene expression, processing, and targeting of active products.

Endocrine cells of the gastric epithelium secrete biologically active peptides and small messenger molecules such as histamine, serotonin, and gamma-aminobutyric acid. The secretory products may act locally (paracrine or autocrine effects) or at distant targets after delivery in the circulation (hormonal effects). the contents of the gastric lumen control both secretion of gastric endocrine cells and the expression of genes involved in the synthesis of their active secretory products; in some cases, gene regulation may occur over periods as short as that required for digestion of a single meal. The conversion of inactive peptide precursors to their active forms takes place during transit along the secretory pathway and is only completed after sequestration in secretory granules. the processing of the gastrin precursor provides a useful model for studying prohormone processing. Generation of the well-known amidated gastrins requires prohormone cleavage and COOH-terminal amidation; the products stimulate acid secretion and mucosal growth. However, recent work indicates that biosynthetic intermediates that do not stimulate acid secretion may nevertheless act at a novel receptor to stimulate growth, so that control of prohormone processing determines which of two alternative types of biologically active peptide is released by gastrin cells. Gastric endocrine cells also have the capacity to accumulate small messenger molecules in secretory vesicles, via proton exchangers. Recent work indicates physiological regulation of the expression of genes encoding cytosolic enzymes such as histidine decarboxylase, which converts histidine to histamine, and of secretory granule transporters such as vesicular monoamine transporter type 2, which concentrates amines in secretory vesicles. Together these findings suggest that modulation of regulatory peptide and amine biosynthesis in gastric endocrine cells constitutes a primary response of the stomach to the arrival of a meal.

Amino Acid Sequence↗

PubMedDate not supplied

Replicative senescence, aging and growth-regulatory transcription factors.

Normal somatic cells invariably enter a state of permanent growth arrest and altered function after a finite number of divisions. This phenomenon is termed cellular or replicative senescence. Replicative senescence is thought to be a tumor-suppressive mechanism, and a contributing factor in aging. Three features distinguish senescent from presenescent cells: an irreversible block to cell proliferation, increased resistance to apoptotic death, and changes in differentiated functions. Senescence entails an altered pattern of gene expression, much of which is due to altered transcription. At least three growth regulatory transcriptional modulators are repressed in senescent cells: the c-fos component of the AP1 transcription factor, the Id1 and Id2 helix-loop-helix (HLH) proteins that negatively regulate basic HLH transcription factors, and the E2F-1 component of the E2F transcription factor. Failure to express any one of these modulators is very likely sufficient to arrest cell proliferation. Loss of these modulators may also explain many of the functional changes shown by senescent cells. In the case of c-fos repression, the resulting decline in AP-1 activity may be exacerbated by an altered ratio of AP-1 components to a protein known as QM or Jif. QM interacts with the c-jun component of AP-1 and suppresses AP-1 activity. We cloned QM from a senescent fibroblast cDNA library, and found that it was neither cell cycle- nor senescence-regulated. However, QM suppressed the growth of murine and human fibroblasts when overexpressed. Thus, an altered balance between positive factors (e.g., AP-1 components) and negative factors (e.g., QM) may lead to the growth arrest, as well as the changes in differentiated gene expression, that are a hallmark of senescent cells.

Carrier Proteins↗

PubMed1980

Defects in enzyme regulation versus defects in enzyme synthesis as cause of metabolic disorders.

Based on the consideration that normal metabolic processes depend upon the activity of key enzymes (and membrane carriers) as modulated by regulatory factors (hormones, diet, endogenous compounds, age, physical activity, environmental agents), metabolic disorders might be classified into two groups: (I) defects in enzyme synthesis, leading to enzyme deficiency (classical inborn errors of metabolism) or to qualitative (structural) enzyme alterations (entailing unresponsiveness to regulation), in the presence of normal regulatory factors; (II) defects in enzyme regulation, which include metabolic syndromes such as diabetes mellitus, obesity and hyperlipoproteinemias (other than type I), and are due to changes in enzyme activities caused by alterations in regulatory factor(s) (secondary to various causes), in the presence of normally responsive enzymes.

Allosteric Regulation↗

PubMed2000

Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation.

BACKGROUND: We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The NO donor diethylenetriamineNONOate (10(-5) to 10(-3) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with N(G)-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10(-3) mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO. CONCLUSIONS: NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2005

Myocardin enhances Smad3-mediated transforming growth factor-beta1 signaling in a CArG box-independent manner: Smad-binding element is an important cis element for SM22alpha transcription in vivo.

Transforming growth factor (TGF)-beta1 is an important cytokine involved in various diseases. However, the molecular mechanism whereby TGF-beta1 signaling modulates the regulatory network for smooth muscle gene transcription remains largely unknown. To address this question, we previously identified a Smad-binding element (SBE) in the SM22alpha promoter as one of the TGF-beta1 response elements. Here, we show that mutation of the SBE reduces the activation potential of a SM22alpha promoter in transgenic mice during embryogenesis. Chromatin immunoprecipitation assays reveal that TGF-beta1 induces Smad3 binding to the SM22alpha promoter in vivo. A multimerized SBE promoter responsive to TGF-beta1 signaling is highly activated by Smad3 but not by the closely related Smad2. Intriguingly, myocardin (Myocd), a known CArG box-dependent serum response factor coactivator, participates in Smad3-mediated TGF-beta1 signaling and synergistically stimulates Smad3-induced SBE promoter activity independent of the CArG box; no such synergy is seen with Smad2. Importantly, Myocd cooperates with Smad3 to activate the wild-type SM22alpha, SM myosin heavy chain, and SMalpha-actin promoters; they also activate the CArG box-mutated SM22alpha promoter as well as the CArG box-independent aortic carboxypeptidase-like protein promoter. Immunopreciptiation assays reveal that Myocd and Smad3 directly interact both in vitro and in vivo. Mutagenesis studies indicate that the C-terminal transactivation domains of Myocd and Smad3 are required for their functional synergy. These results reveal a novel regulatory mechanism whereby Myocd participates in TGF-beta1 signal pathway through direct interaction with Smad3, which binds to the SBEs. This is the first demonstration that Myocd can act as a transcriptional coactivator of the smooth muscle regulatory network in a CArG box-independent manner.

Animals↗

PubMed1990

Beta-adrenergic regulation of the muscarinic-gated K+ channel via cyclic AMP-dependent protein kinase in atrial cells.

Cholinergic and beta-adrenergic stimulations of ionic currents are major physiological mechanisms in the regulation of heart rate and contractility. Muscarinic receptor stimulation is known to reduce beta-adrenergic effects on calcium current via reduction of cyclic AMP. Whether the beta-adrenergic stimulation affects the muscarinic response is not known. I report here that the beta-adrenergic agonist isoproterenol enhanced the muscarinic-activated K+ channel activity in rat atrial cells. Application of cyclic AMP-dependent protein kinase or its catalytic subunit to the cytoplasmic side of the membrane augmented the acetylcholine-activated K+ channel activity twofold to threefold. Increases in channel activity produced by isoproterenol or cyclic AMP-dependent protein kinase were associated with fourfold to fivefold and approximately twofold increases in the mean open and closed time durations, respectively. Alkaline phosphatase treatment reversed these effects. These results suggest that cyclic AMP-dependent phosphorylation of the K+ channel or associated regulatory proteins modulates the gating kinetics of the channel. This mechanism may be important in the regulation of pacemaker activity and, thus, the heart rate during beta-adrenergic stimulation.

Alkaline Phosphatase↗

PubMed2002

Noncoding RNA danger motifs bridge innate and adaptive immunity and are potent adjuvants for vaccination.

The adaptive immune response is triggered by recognition of T and B cell epitopes and is influenced by "danger" motifs that act via innate immune receptors. This study shows that motifs associated with noncoding RNA are essential features in the immune response reminiscent of viral infection, mediating rapid induction of proinflammatory chemokine expression, recruitment and activation of antigen-presenting cells, modulation of regulatory cytokines, subsequent differentiation of Th1 cells, isotype switching, and stimulation of cross-priming. The heterogeneity of RNA-associated motifs results in differential binding to cellular receptors, and specifically impacts the immune profile. Naturally occurring double-stranded RNA (dsRNA) triggered activation of dendritic cells and enhancement of specific immunity, similar to selected synthetic dsRNA motifs. Based on the ability of specific RNA motifs to block tolerance induction and effectively organize the immune defense during viral infection, we conclude that such RNA species are potent danger motifs. We also demonstrate the feasibility of using selected RNA motifs as adjuvants in the context of novel aerosol carriers for optimizing the immune response to subunit vaccines. In conclusion, RNA-associated motifs produced during viral infection bridge the early response with the late adaptive phase, regulating the activation and differentiation of antigen-specific B and T cells, in addition to a short-term impact on innate immunity.

Adaptation, Physiological↗

PubMed2002

A polyaromatic caveolin-binding-like motif in the cytoplasmic tail of the type 1 receptor for angiotensin II plays an important role in receptor trafficking and signaling.

The type 1 receptor for angiotensin II (AT(1)) is a member of the G protein-coupled receptor family. The presence of a caveolin-binding-like motif (phiXphiXXXXphiXXphi where phi is an aromatic residue) within the cytoplasmic tail of the AT(1) receptor suggests an implication for caveolae in the functionality of this receptor. We constructed a mutant AT(1) receptor where each of the aromatic residues in the caveolin-binding-like motif were replaced by alanine (AT(1)-YFFY/A). Mutation of this motif considerably reduced the plasma membrane expression of the receptor that accumulated in a perinuclear compartment. The agonist-induced internalization rate of the AT(1)-YFFY/A receptor was also significantly reduced. Finally, the AT(1)-YFFY/A receptor was poorly activated as indicated by a low agonist-induced production of inositol phosphates. Unexpectedly, the proportion of AT(1) receptor found in caveolae was minor under basal conditions and did not increase under stimulated conditions. Coexpression of the AT(1) receptor with dopamine receptor interacting protein of 78 kDa, a protein implicated in the cellular routing of the dopamine D1 receptor, increased plasma membrane expression of the AT(1) receptor. However, dopamine receptor interacting protein of 78 kDa had no effect on the expression of the AT(1)-YFFY/A receptor. Taken together, these results suggest that the caveolin-binding-like motif of the AT(1) receptor does not promote localization of the receptor to caveolae but rather may act as a docking site for regulatory proteins modulating the routing and the functionality of the receptor.

Angiotensin II↗

PubMed2003

Biological activity of activating thyroid-stimulating hormone receptor mutants depends on the cellular context.

Activating TSH receptor (TSHR) mutations are a major cause of toxic thyroid adenoma and familial hyperthyroidism, and more than 37 such mutations have been described. Previously their functional activity had been assessed in terms of cAMP and inositol phosphate production and predominantly in transiently transfected COS-7 (monkey embryonic kidney cells), a model that does not reflect effects on thyrocyte proliferation and function. Here we have performed a systematic comparison of wild-type and seven gain-of-function TSHR mutants, introduced into rat FRTL-5 and human thyrocytes, using retroviral vectors. Our results show that 1) biological potency of TSHR mutants in thyroid cells does not correlate with their cAMP levels in transfected COS cells, highlighting the importance of cellular context and level of expression when assessing biological effects of oncogenic mutations; 2) dissociation between stimulation of function and growth occurs with thyrocyte differentiated functions more readily stimulated than growth; 3) TSHR mutants show a similar order of potency in FRTL-5 cells and human thyrocytes; 4) mutants inducing the highest stimulation of adenylyl cyclase may paradoxically fail to induce proliferation; and 5) biological effects of cAMP activating TSHR mutants are attenuated by complex counterregulatory mechanisms at least at the level of phosphodiesterases and cAMP regulatory element modulator isoforms.

Amino Acid Sequence↗

PubMed1980

Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro.

Acetylcholine, at concentrations of 10(-10)--10(-7) M, inhibited the release of immunoreactive somatostatin (SRIF) from rat hypothalamic segments which had been maintained in short term culture for 24 h. Neostigmine (10(-6) M), an anticholinesterase, also inhibited the release of SRIF, whereas atropine (10(-6) M), a muscarinic anticholinergic, had no effect on basal SRIF release but blocked the inhibition caused by acetylcholine (10(-8) M). However, hexamethonium (10(-6) M), a nicotinic antagonist, did not abolish the inhibition induced by acetylcholine. Potassium depolarization (56 mM KCl) caused stimulation of SRIF release, which was dependent on the presence of calcium in the incubation medium. SRIF was measured by a RIA sensitive to 1 pg/tube. Authenticity of immunoreactive SRIF released was suggested by immunological parallelism and chromatographic criteria using gel and high pressure liquid systems. These results suggest that muscarinic cholinergic mechanisms may have a regulatory role modulating the secretion of SRIF and, consequently, GH through actions at a hypothalamic level.

Acetylcholine↗

PubMed1990

The androgen-binding protein gene is expressed in male and female rat brain.

Extracellular androgen-binding proteins (ABP) are thought to modulate the regulatory functions of androgens and the trans-acting nuclear androgen receptor. Testicular ABP and plasma sex hormone-binding globulin (SHBG), which is produced in liver, are encoded by the same gene. We have now found that the ABP-SHBG gene is also expressed in male and female rat brain. Immunoreactive ABP was found to be present in neuronal cell bodies throughout the brain as well as in fibers of the hypothalamic median eminence. The highest concentrations of immunoreactive cell bodies were located in the supraoptic and paraventricular nuclei. Likewise, ABP mRNA was present in all brain regions examined. Analysis of cDNA clones representing brain ABP mRNAs revealed amino acid sequence differences in brain and testicular ABPs. The protein encoded by an alternatively processed RNA has sequence characteristics suggesting that the protein could act as a competitior of ABP binding to cell surface receptors. These data and gene-sequencing experiments indicate that a specific ABP gene promoter is used for transcription initiation in brain. ABP may function in brain as an androgen carrier protein; however, in view of the widespread presence of ABP and ABP mRNA in brain, the protein may have a much broader, yet unknown, function.

Amino Acid Sequence↗