Regulation Of Specific Gene Expression Research Articles

Nuclear matrix and the regulation of gene expression: tissue specificity.

Tissue specific regulation of gene expression by a single transcription factor or group of transcription factors cannot be explained simply by DNA sequence alone. For example, in the same animal a particular transcription factor is capable of interacting with DNA in the nucleus of many different cell types, resulting in unique gene expressions despite the presence of a similar genome in all cells. Historically, these differences in response to a single type of factor within target tissues in the same animal have been suggested to occur through different alterations in chromatin structure. Recent, data has demonstrated that combinations of hormones and transcription factors working together may cooperatively play a role in the regulation of gene expression [Pearce and Yamamoto (1993): Science 259:1161-1165]. However, the molecular mechanisms of this tissue specific regulation of gene expression still remains largely unexplained. Current evidence suggests that in different cell types the interplay between the specific three-dimensional organization of the genome and the structural components of the nucleus, the nuclear matrix, may accomplish the regulation of specific gene expression.

ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation.

DNA-binding proteins containing the basic helix-loop-helix (bHLH) domain have been implicated in lineage determination and the regulation of specific gene expression in a number of cell types. By oligonucleotide screening of an adipocyte cDNA expression library, we have identified a novel member of the bHLH-leucine zipper transcription factor family designated ADD1. ADD1 mRNA is expressed predominantly in brown adipose tissue in vivo and is regulated during both determination and differentiation of cultured adipocyte cell lines. ADD1 can function as a sequence-specific transcriptional activator in that it stimulates expression of a chloramphenicol acetyltransferase vector containing multiple ADD1 binding sequences but is unable to activate the myosin light-chain enhancer, which contains multiple binding sites for another bHLH factor, MyoD. ADD1 can also activate transcription through a binding site present in the 5'-flanking region of the fatty acid synthetase gene which is expressed in a differentiation-dependent manner in adipose cells. These data suggest that ADD1 plays a role in the regulation of determination- and differentiation-specific gene expression in adipocytes.

Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL.

The regulation of specific gene expression by nitrate in Escherichia coli is mediated by the NarX/NarQ-NarL system. Based on sequence homologies with a family of two-component regulatory systems in bacteria, NarL has been identified as a putative response regulator while NarX and NarQ were proposed to be alternative membrane-associated sensors that activate NarL in the presence of nitrate. To investigate the interaction of NarX and NarL in vitro, both proteins were purified from overproducing strains. Purified NarX was rapidly labeled when incubated with [gamma-32P] ATP but not with [alpha-32P]ATP in a reaction that required Mg2+ but was unaffected by nitrate. Incubation of the labeled NarX with purified NarL resulted in the transient phosphorylation of NarL. Both the phosphorylation and dephosphorylation of NarL required Mg2+, and neither reaction was affected by the presence of nitrate. NarL-phosphate, stabilized by the addition of EDTA, ran as a monomer on gel filtration. Dephosphorylation of the isolated NarL-phosphate required the addition of both Mg2+ and the NarX protein. The relative stabilities of the phosphorylated forms of the two proteins at different pH values were consistent with the proposal that, in analogy to other related two-component regulatory systems, NarX and NarL were phosphorylated on specific histidine and aspartate residues, respectively.

Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides

Publisher Summary This chapter discusses the synthetic nucleic acids interfering with gene expression (SNAIGE), which are being developed as specific regulators of gene expression both for fundamental studies and/or for potential (medical) applications. Although straightforward in principle, the approach still faces many unknowns. Cell-free studies with model systems have helped one to learn much about the various modes through which oligonucleotides bind to their nucleic acids or protein targets. Increasing numbers of modifications allowing improved resistance toward nucleases or more efficient interaction with various targets have been devised and more will be forthcoming. A most exciting prospect has been enabled by synthetic material; the normal backbone of DNA has been replaced by a polyamide linkage, giving rise to “polyamide nucleic acid” that retains specificity in base recognition. Although a large number of genes have been successfully down-regulated in various biological systems. The tools of cell biology will help in solving pending questions, such as intracellular routing of SNAIGE, involvement of RNase H in their action, and accessibility of various sites in mRNAs, pre-mRNAs, or DNAs in their cellular environment. This helps to devise new tools on a more rational basis and to choose the best strategy in each case.

Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.

Inhibition of c-Ha-ras gene expression by hammerhead ribozymes containing a stable C(UUCG)G hairpin loop.

Catalytic RNAs recognize specific sequences of RNA and cleave at a specific site. In this study, we designed hammerhead ribozymes with a thermodynamically stable loop of the sequence 5'C(UUCG)G3' to prevent the aggregation of ribozymes with hammerhead structures. The cleavage activities of these ribozymes were examined using a synthetic pentadecamer with the sequence for the c-Ha-ras mRNA mutated at codon 12 (GGU-->GUU). For in vivo studies, we constructed a plasmid which expressed a highly active ribozyme targeted against the mutated c-Ha-ras mRNA. When this ribozyme-encoding gene and the activated c-Ha-ras gene were cotransfected into NIH3T3 cells, morphologically normal cells were obtained. We also determined that the expression of the c-Ha-ras gene was inhibited in these cells. These results show that ribozymes containing this stable hairpin loop are useful for the regulation of specific gene expression in vivo.

Glucose regulation of specific gene expression is altered in a glucokinase-deficient mutant of Tetrahymena

Expression of the galactokinase gene in Tetrahymena thermophila can be repressed by glucose, glucose analogs, and epinephrine, each apparently acting through increased intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) (1). To characterize further the initial steps in the control of galactokinase gene expression by glucose, we have analyzed mutants which are defective in the metabolism of this sugar; these mutants were selected for their resistance to the glucose analog, 2-deoxyglucose (2). In one such mutant that is deficient in glucokinase, the synthesis of galactokinase is totally resistant to repression by glucose or its analogs, while repression by exogenous catecholamines or dibutyryl cAMP is unaffected. Radiochromatographic analyses of extracts of wild-type cells incubated with [14C]-deoxyglucose reveal intracellular conversion to several deoxyglucose metabolites, principally deoxyglucose-6-P and smaller amounts of deoxyglucose-1-P and 2-deoxygluconate; extracts of glucokinase-deficient cells prepared in a similar manner contain only trace amounts of deoxyglucose-6-P. The glucose analog 3-O-methylglucose, which is transported but not phosphorylated in wild-type cells, also cannot maintain repression of galactokinase. These results establish that the transport and subsequent phosphorylation of glucose are required for glucose-initiated repression of galactokinase gene expression, possibly acting by modulation of catecholamine or cyclic AMP levels. Additionally, we show unequivocally that: (a) cells containing derepressed levels of galactokinase are repressed upon the addition of glucose by inhibition of the synthesis of new enzyme and dilution of preformed enzyme concomitant with cell division, rather than through selective inactivation or degradation of galactokinase; and (b) glycerol kinase, glucokinase and fructokinase activities also are repressed by glucose in wild-type Tetrahymena, indicating that the glucose repression phenomenon is pleiotropic. Because the glucose repression of the synthesis of each of these enzymes is abolished in cells deficient in glucokinase, the regulatory mechanisms elucidated for repression of galactokinase synthesis are likely to be of wide significance.

Poly(ADP-Ribose) Polymerase Inhibitors Suppress UV-Induced Human Immunodeficiency Virus Type 1 Gene Expression at the Posttranscriptional Level

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.

Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level.

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.

The Tissue Matrix: Cell Dynamics and Hormone Action*

THE SPECIFIC response of individual tissues to a single steroid receptor cannot be explained simply by DNA sequence alone. For example, in the same animal a given steroid receptor is capable of interacting with the nucleus of two different cell types, resulting in unique gene expressions despite the presence of a similar genome. Historically, these differences in response to a single type of steroid receptor within target tissues in the same animal have been suggested to occur through different alterations in chromatin structure. However, the molecular mechanisms of this tissue specificity remain unexplained. It is possible that in different cell types the specific three-dimensional organization of the genome and cell skeletal elements interplay may vary to accomplish the hormonal regulation of specific gene expression. It is the purpose of this review to address the potential role of cell structure as a central component of hormone action.

Cooperative stimulation of specific gene transcription by epidermal growth factor and transforming growth factor type beta 1.

Transforming growth factor type beta 1 (TGF-beta 1) is a pleiotropic regulator of cell growth and differentiation which can potentiate or otherwise modify cellular responses to different growth factors such as epidermal growth factor (EGF). Since cellular responses to peptide growth factors are mediated, in part, through the regulation of specific gene expression, we have studied the effect of TGF-beta 1 on the EGF-dependent transcriptional of a diverse panel of genes which included the fibronectin gene, the cytoskeletal beta- and gamma-actin genes, and the c-fos protooncogene. The addition of a combination of TGF-beta 1 (10 ng/ml) and EGF (10 ng/ml) to quiescent AKR-2B cells maintained in serum-free medium resulted in a strong synergistic stimulation of transcription which was not evident using either growth factor individually at concentrations of 20-100 ng/ml. This effect was evident within 10 min and did not require the continual presence of TGF-beta 1 in the culture medium. Increased responsiveness of some gene promoters to EGF stimulation was evident for up to 6 h following a brief 10-min exposure to TGF-beta 1. Similar exposure to either platelet-derived growth factor or fibroblast growth factor had little or no effect on EGF-stimulated transcription. These results indicate that the transcriptional response of specific genes to EGF can be stably altered as a consequence of exposure to TGF-beta 1.

Phenobarbital-inducible Aldehyde Dehydrogenase in the Rat: cDNA sequence and regulation of the mRNA by phenobarbital in responsive rats

Abstract In the rat, a cytosolic isozyme of aldehyde dehydrogenase, designated ALDH-PB, can be induced in the liver by administration of phenobarbital (PB). ALDH-PB activity and mRNA are induced in Long-Evans rats that possess a responsive (R) allele but are not induced in homozygous nonresponsive rats (rr), although the rr genotype is competent to induce other PB-responsive mRNAs. ALDH-PB mRNA is expressed in the basal state (without PB administration) in hepatic tissue in both RR and rr genotypes. We report the complete nucleotide sequence of the rat ALDH-PB mRNA. The protein encoded by the ALDH-PB mRNA is 501 amino acids in length and has a predicted molecular mass of 54,540 daltons. The amino acid sequence predicted from the mRNA demonstrates a strong conservation between the rat ALDH-PB and the human cytosolic aldehyde dehydrogenase hALDH-1. We demonstrate the ALDH-PB, cytochrome P-450b, cytochrome P-450e, and glutathione S-transferase Ya subunit mRNA levels in the liver are altered noncoordinately by administration of PB in RR and rr genotypes. The strikingly different responses to PB administration between the various mRNA species in each of the genotypes suggest that the regulation of specific gene expression by PB may involve multiple pathways.

Regulation of the synthesis of steroidogenic enzymes in adrenal cortical cells by ACTH.

The mechanism of steroid hormone regulation of specific gene expression in target cells has provided a major focus of interest for investigators concerned with molecular mechanisms involved in cell regulation. This work has been given renewed impetus by the recent cloning and characterization of several steroid hormone receptors (1-6), the trans-acting elements which, upon binding specific ligands, namely the steroid hormones, interact with specific regions of many eukaryotic genes to alter their expression. In contrast, there has been considerably less interest in the mechanisms of regulation of steroid hormone biosynthesis. Yet this is also a topic of great importance, not only because these reactions provide the ligands that interact with the steroid hormone receptors, but also because the steroidogenic enzymes provide excellent model systems for studying the regulation of gene expression by cAMP-dependent mechanisms and by mechanisms involving protein kinase C and tyrosine kinase activity. A variety of mechanisms appear to regulate steroid hydroxylase gene expression in a given cell, and tissue-specific as well as developmental factors play important roles also. In this review, we focus primarily on the regulation of steroid hydroxylase gene expression by cAMP­ dependent mechanisms and, in particular, the mechanisms of ACTH regula­ tion of the synthesis of the steroidogenic enzymes present in the adrenal cortex.

Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase mRNA and structures of the 5' and 3' flanking regions of the serine dehydratase gene.

Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.

Changing patterns of transcriptional and post-transcriptional control of liver-specific gene expression during rat development.

Genes coding for unique or tissue-specific (differentiated) functions in the liver are induced at different times during development. It has generally been felt that transcriptional control represents the dominant mechanism for regulating expression of these genes. We have determined the relative transcription rates and mRNA steady-state levels for a series of genes specifically or preferentially expressed in rat liver and find examples of transcriptional control (albumin, alpha-fetoprotein, alpha 1-antitrypsin, tyrosine aminotransferase, transferrin, and cytochrome P450, TF-1) and post-transcriptional control (alpha 1-acid glycoprotein, apolipoproteins A-1 and E, malic enzyme, and ATP citrate lyase), as well as "mixed" regulation (ligandin and cytochrome P450, R17). Examples have been identified in which the predominant mode for regulating expression of preferentially expressed genes changes from transcriptional to post-transcriptional at different stages of liver development and some members of multigene families (cytochrome P450s and apolipoprotein genes) also show independent and sometimes contrasting modes of regulation. Therefore, it appears that regulation of specific gene expression in the liver is a dynamic process, far more complex than heretofore suspected, and a much greater contribution of post-transcriptional regulation accounts for changes in expression of genes representing major functions of the liver.

49] Ca2+/Calmodulin regulation of prolactin gene expression

Despite the extensive literature on the biological actions of Ca2+ and calmodulin, very little is known about their involvement in nuclear functions, e.g., regulation of specific gene expression. To date, the only genes other than prolactin and growth hormone shown to be regulated by perturbations in cell Ca2+ are those coding for two glucose-regulated proteins. However, there is a growing body of indirect evidence for nuclear functions of Ca2+ and calmodulin, and we suspect that other examples of Ca2+-regulated genes will emerge. We have described in this chapter several different experimental approaches which we have employed to examine first whether prolactin gene expression is regulated by changes in cell Ca2+ content, and then to begin searching for the components of the mechanism by which Ca2+ exerts its effects on the prolactin gene. The tentative identification of 56-kDa nuclear matrix protein as both a calmodulin-binding protein and a substrate of a Ca2+-calmodulin-dependent protein kinase suggests that NMP 56 may be a subunit of a multifunctional Ca2+-calmodulin-protein kinase. This enzyme was recently detected in the nuclear matrix fraction of neuronal nuclei, and was shown to phosphorylate a chromatin protein similar to high mobility group protein 17 (HMG 17). Since HMG 17 is associated with actively transcribed chromatin, its phosphorylation in GH3 cells might play a role in the Ca2+-calmodulin-dependent regulation of prolactin gene expression by hormones and growth factors.

Differential regulation of specific gene expression in mouse kidney by androgens and antiandrogens.

Androgens, as other steroid hormones, interact with a receptor protein to regulate gene expression in target tissues.1 Much of the work to date on this subject has involved analysis of the abundant gene products stimulated by androgens in the rat prostate2,3 and seminal vesicle.4–6 However, no attempts were made in these studies to correlate specific gene expression with androgen receptor dynamics or to compare the androgen sensitivity of these different genes in male accessory sex organs.KeywordsAndrogen ReceptorOrnithine DecarboxylaseMouse KidneymRNA ConcentrationReceptor ConcentrationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Influence of pyridoxal 5'-phosphate on the DNA binding activity of steroid hormone receptors and other DNA binding proteins

1. introduction The interaction of steroid hormone receptors with the genome of the target cells has attracted much at- tention. This interaction is considered responsible for the regulation of specific gene expression by steroid hormones. In [ 1,2] pyridoxal5’.phosphate was ob- served to inhibit the binding of steroid hormone recep- tors to immobilized DNA or nucleotides [ 1,2]. Pyri- doxal phosphate may also operate in vivo modulating the interaction of the glucocorticoid receptor with the hepatic cell nuclei [3]. The 4’.aldehyde group of pyridoxal5’.phosphate was shown to be required for the interaction with the glucocorticoid receptor [4]. Reduction with NaBH4 leads to irreversible loss of DNA binding activity, suggesting the formation of a Schiff base intermediate [5]. Very little is known, however, on the protein spec- ificity of this pyridoxal phosphate effect. In fact pyridoxal5’.phosphate could have a general inhibitory effect on DNA binding proteins simply due to its abil- ity to react with amino groups in the active site, thus interferring with electrostatic interactions. To explore this question we have investigated the influence of pyridoxal phosphate on DNA binding activity of puri- fied hepatic glucocorticoid receptor [6], of the

Biochemical homology between rat dorsal prostate and coagulating gland. Purification of a major androgen-induced protein.

The anatomically distinct organs, rat dorsal prostate and coagulating gland, were found to display remarkable homology in protein composition, including two major androgen-dependent secretory proteins, referred to as dorsal proteins I and II. Dorsal protein I has been purified and found to be a dimer composed of two identical subunits with sedimentation coefficient 4.6 S, Stokes radium 32 A, and Mr = 71,000 (62,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis). This dimer (6.8 S, 46 A, Mr = 150,000) dissociates at high ionic strength and can be partially reconstituted by removal of salt. Dorsal protein I is a basic protein (pI 9) with high lysine content and binds to phosphocellulose but not to DEAE-Sepharose. Schiff's staining shows that it contains carbohydrate. Quantitative rocket immunoelectrophoresis using a rabbit antiserum indicates dorsal protein I is produced only in dorsal prostate and coagulating gland. The protein constitutes approximately 25% of total cytosol protein in both organs, yet makes up on 5% of coagulating gland luminal fluid and ejaculated seminal fluid. It was not detected in the rat dorsal prostate tumor (Dunning R3327H). Dorsal protein II is a larger protein (Mr = 80,000 by SDS gel electrophoresis) with higher carbohydrate content. Under nondenaturing conditions, it has a Stokes radius of > 200 A, corresponding to Mr of > 300,000. Dorsal protein II represents a smaller proportion of total cytosol protein than does dorsal protein I, yet is the predominant protein of coagulating gland fluid. The proteins will be useful probes for studies on androgen regulation of specific gene expression and prostatic secretory mechanisms.

The Differential Stimulatory Effect of Thyroid Hormone on Growth Hormone Synthesis and Estrogen on Prolactin Synthesis due to Accumulation of Specific Messenger Ribonucleic Acids*

Thyroid and steroid hormones bind to chromatin-associated nuclear protein receptors which appear to be hormone specific and are believed to mediate the action of these hormones through synthesis of mRNA. The purpose of this investigation was to test the hypothesis of hormonal control through regulation of specific gene expression. Rat GH and PRL were chosen as a model, since previous work has indicated that the synthesis of these hormones is regulated by thyroid hormone and estrogen, respectively. Male rats were kept intact (N) or were thyroidectomized (Tx) and treated with estradiol (N + E or Tx + E) or replaced with T4, (Tx + T4). GH and PRL synthesis was determined in quarter pituitaries by the in vitro incorporation of [3H]leucine into specific immunoprecipitates. GH and PRL mRNA activity was measured by cell-free translation of total RNA extracted from the remaining pituitary tissue. Tx suppressed both GH synthesis and mRNA activity to 2.8% and 1.4% of the level in N rats and was not affected by trea...