Nuclear Run-off Transcription Assays Research Articles

The chicken skeletal alpha-actin gene promoter region exhibits partial dyad symmetry and a capacity to drive bidirectional transcription.

The chicken skeletal alpha-actin gene promoter region (-202 to -12) provides myogenic transcriptional specificity. This promoter contains partial dyad symmetry about an axis at nucleotide -108 and in transfection experiments is capable of directing transcription in a bidirectional manner. At least three different transcription initiation start sites, oriented toward upstream sequences, were mapped 25 to 30 base pairs from TATA-like regions. The opposing transcriptional activity was potentiated upon the deletion of sequences proximal to the alpha-actin transcription start site. Thus, sequences which serve to position RNA polymerase for alpha-actin transcription may allow, in their absence, the selection of alternative and reverse-oriented start sites. Nuclear runoff transcription assays of embryonic muscle indicated that divergent transcription may occur in vivo but with rapid turnover of nuclear transcripts. Divergent transcriptional activity enabled us to define the 3' regulatory boundary of the skeletal alpha-actin promoter which retains a high level of myogenic transcriptional activity. The 3' regulatory border was detected when serial 3' deletions bisected the element (-91 CCAAA TATGG -82) which reduced transcriptional activity by 80%. Previously we showed that disruption of its upstream counterpart (-127 CCAAAGAAGG -136) resulted in about a 90% decrease in activity. These element pairs, which we describe as CCAAT box-associated repeats, are conserved in all sequenced vertebrate sarcomeric actin genes and may act in a cooperative manner to facilitate transcription in myogenic cells.

Beta-adrenergic receptors in hamster smooth muscle cells are transcriptionally regulated by glucocorticoids.

Steroid hormones modulate adrenergic receptor responsiveness and receptor number. To investigate the regulation of the beta 2-adrenergic receptor gene by glucocorticoids we examined the effects of the synthetic glucocorticoid agonist triamcinolone acetonide on the expression of beta 2-adrenergic receptors in DDT1MF-2 hamster smooth muscle cells. Glucocorticoid treatment (1 X 10(-7) M) produced a 2.2 +/- 0.4-fold (n = 8) increase in beta 2-adrenergic receptor number (maximum) between 6 and 12 h) as determined by radioligand binding and a similar increase in catecholamine-stimulated adenylate cyclase activity. Steady-state levels of beta 2-adrenergic receptor mRNA, analyzed by Northern blot hybridization, were increased 2.4 +/- 0.4-fold (n = 6) within 1 h, while actin mRNA levels were unchanged throughout the experiment. These steroid-induced increases in beta 2-adrenergic receptor mRNA returned to control levels by 24 h and were followed by a much slower decline in beta 2-adrenergic receptor in plasma membranes. The rate of beta 2-adrenergic receptor gene transcription, assessed by nuclear run-off transcription assays, increased 3.1 +/- 0.1-fold (n = 2) in cells treated for 30 min with 1 X 10(-7) M triamcinolone acetonide. These studies indicate that glucocorticoids regulate the beta 2-adrenergic receptor-adenylate cyclase system by controlling the rate of transcription of the beta 2-adrenergic receptor gene and hence the responsiveness of the enzyme to catecholamine stimulation.

Transcriptional activation of HLA-DR alpha by interferon gamma requires a trans-acting protein.

Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-gamma-induced accumulation of HLA-DR alpha mRNA, indicating that de novo synthesis of a trans-acting protein factor is required for induction of this major histocompatibility complex class II gene. Nuclear run-off transcription assays revealed that IFN-gamma acts by directly stimulating the transcription rate of HLA-DR alpha. Similarly, IFN-gamma increased the transcription rate of the class I HLA-A2-encoding gene as well as that of the human invariant chain gene. IFN-gamma-induced transcription of HLA-DR alpha and of the invariant chain gene was blocked by treatment with cycloheximide, but IFN-gamma-induced transcription of HLA-A2 was unaffected. Our findings show that transcriptional induction of HLA-DR alpha and the invariant chain gene by IFN-gamma requires the action of an unidentified trans-acting protein.

Changes in heat shock protein synthesis and hsp70 gene transcription during erythropoiesis of Xenopus laevis

As erythroid cells of Xenopus laevis differentiate, their capacity for transcription and protein synthesis becomes progressively restricted. Mature erythrocytes are thought to be transcriptionally inert and have very low levels of protein synthesis ( Maclean et al., 1973 , Cell Differ. 2, 261–269). We have examined actual and potential synthetic activity during erythropoiesis by comparing the patterns of protein synthesis and measuring the transcriptional activity of selected genes under ambient and heat shock conditions. As erythropoiesis proceeds, there is a progressive decrease in the number of heat shock proteins (hsps) whose synthesis is induced at elevated temperatures. Despite this repression, mature erythrocytes are able to respond to heat shock with the induction of synthesis of one protein, hsp70. In addition, an hsp70-like protein is synthesized constitutively in orthochromatic erythroblasts, the final erythroid cell stage before maturation into erythrocytes. An in vitro nuclear run-off transcription assay was used to study transcription of the hsp70, β-globin, and ribosomal genes during erythropoiesis. β-Globin and ribosomal gene transcription were detected at all stages of erythropoiesis, including the erythrocyte stage. However, transcription of the β-globin gene relative to ribosomal gene transcription declines substantially in erythrocytes as compared to earlier stages. Hsp70 gene transcription shows both stage-specific and heat shock-dependent transcription. Orthochromatic erythroblasts and erythrocytes have heat shock-dependent hsp70 gene transcription, whereas the gene is transcribed at the earlier erythroblast stages at ambient temperature as well as during heat shock. The constitutive hsp70 transcripts made during these earlier stages may be stored for utilization in constitutive synthesis of the hsp70-like protein in orthochromatic erythroblasts. These data indicate that although erythrocytes become synthetically repressed during differentiation, they are less inert than previously thought. The data also reveal that the synthesis of hsps and related proteins is subject to both transcriptional and post-transcriptional control during erythropoiesis.

Inhibition of Transcription of Herpes Simplex Virus Immediate Early Genes in Interferon-treated Human Cells

The effect of interferon (IFN) treatment on the early stages of herpes simplex virus type 1 (HSV-1) replication in three types of human cells was investigated. Interferon pretreatment was shown to reduce the steady state levels of both total and polysomebound HSV-1 immediate early alpha mRNAs. Using the nuclear run-off transcription assay, we showed that IFN selectively inhibited transcription of the HSV-1 genes, with no effect on transcription of total cellular RNA or that of the beta-tubulin RNA. Thus, IFN appears to inhibit the initiation of HSV-1 alpha gene transcription rather than affect the stability of the respective mRNAs. IFN did not prevent the HSV-1-induced early shut-off of host cellular protein synthesis caused by a structural protein of infecting virus. This observation indicated that the IFN-mediated inhibition of HSV-1 replication is at a stage beyond viral penetration into the cytoplasm. These results suggested that IFN blocked HSV-1 replication primarily at a very early stage, during the onset of alpha mRNA transcription.

Involvement of protein kinase C in the transcriptional regulation of ornithine decarboxylase gene expression by 12- O-tetradecanoylphorbol-13-acetate in T24 human bladder carcinoma cells

We have analyzed the molecular mechanisms involved in ornithine decarboxylase (ODC) induction by the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) in T24 cells, an easily manipulable human epithelial cell line. The addition of as low as 10 −9 m TPA to T24 cells, cultured in a serum-free medium, resulted in ODC induction, with peak ODC activity occurring at about 6 h after TPA treatment. The induction of ODC activity correlates with the steady-state levels of ODC mRNA increased by TPA in T24 cells. TPA treatment did not elicit any change in the size (2.1 kb) of ODC mRNA as determined by Northern blot analysis by hybridization to nick-translated 32P-labeled cDNA prepared from either mouse or human ODC mRNA. Using the DNA-excess filter hybridization technique, we found that increased steady-state levels of ODC mRNA after TPA treatment may be the result of enhanced accumulation of newly synthesized ODC mRNA. The magnitude of the induction of ODC activity was proportional to the amount of ODC mRNA synthesis caused by TPA. In a pulse-chase experiment, we failed to detect any difference in the half-life of ODC mRNA in T24 cells after treatment with either TPA or the vehicle ethanol; the half-life of ODC mRNA was about 6 h in both cases. Furthermore, as determined by the “nuclear runoff transcription assay,” the rate of transcription of ODC-gene was increased by treatment of T24 cells with TPA. These results provide direct evidence of the role of transcription-initiation in ODC-gene expression. The examination of the role of protein kinase C in ODC-gene transcription revealed that TPA or diacylglycerol did not induce the synthesis of ODC mRNA in PKC-deficient T24 cells. Taken together these results indicate that the TPA-increased synthesis of steady-state levels of ODC mRNA in T24 cells may be mediated by protein kinase C and is regulated at the transcriptional level.

The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells.

NIH 3T3 cells transformed with the human c-Ha-rasVal-12 oncogene showed markedly enhanced activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, as compared with their nontransformed counterparts. While in normal and in c-Ha-ras proto-oncogene-transfected cells stimulation with serum caused a transient induction of ODC, in cells transfected with the mutant c-Ha-ras oncogene the activity of ODC persisted at high levels for greatly extended periods of time. The amounts of immunoreactive ODC protein roughly paralleled the changes in the enzyme activity. The augmentation of ODC content by transformation could be largely, but not solely, accounted for by an enhanced accumulation of ODC mRNA. Nuclear run-off transcription assays demonstrated that in transformed cells the rate of transcription of the ODC gene was increased but to a much lower extent than the increase in the level of ODC mRNA. The turnover of ODC mRNA, as measured after actinomycin D treatment, was negligible in transformed cells for up to 8 h, whereas in normal cells the messenger content was initially decreased, by 40% within 4 h, and then remained constant. In normal cells, however, actinomycin D depressed the expression of ODC by more than 80%, while in transformed cells the activity of ODC was slightly superinduced, corresponding to the changes of ODC mRNA. These findings suggest that labile proteins may be involved in the regulation of both the stability and translatability of the ODC mRNA. Transformation led also to about 3-fold stabilization of ODC as determined by an exposure of the cells to cycloheximide. The results thus suggest ODC deregulation at multiple levels in the ras-oncogene-transformed cells.

Cloning of glucocorticoid-responsive mRNA in the rat thymus.

To elucidate the molecular mechanism of rat thymus involution induced by administration of glucocorticoid, we screened a cDNA library for polysomal poly(A)+RNA from adrenalectomized rat thymi by a differential colony hybridization method. Labeled cDNAs for mRNAs isolated from thymi of adrenalectomized rats and rats receiving dexamethasone (Dex) treatment were used as probes. Eight cDNA clones for mRNAs which had a diminished response to administration of Dex were isolated. The relative concentration of cloned mRNAs in the thymic polysomal RNA was significantly decreased 3 h after hormone administration, while changes in the nuclear RNA were not significant after Dex administration. One of the selected cDNA clones designated pRTGR-8 corresponded to mRNA of about 3,000 nucleotides, and a nuclear run-off transcription assay indicated that the rate of transcription of pRTGR-8 RNA was repressed by Dex administration. The cloned cDNAs obtained in this experiment may provide useful probes for studying the negative regulation mechanism of gene expression by glucocorticoids.

Complex regulation of the muscle-specific contractile protein (troponin I) gene.

A cloned quail troponin I contractile protein gene, stably transfected into a mouse myogenic cell line, exhibits appropriate developmental activation and quantitative expression during myoblast differentiation. Deletion mutagenesis analyses reveal that the troponin I gene has two distinct cis regulatory elements required for its developmental expression, as measured by mRNA accumulation and nuclear runoff transcription assays. One element in the 5' flanking region is required for maximum quantitative expression, and a second larger regulatory element (1.5 kilobases) within the first intron is responsible for differentiation-specific transcription. The upstream region is highly sensitive to negative repression by interaction with pBR322 sequences. The larger intragenic region retains some activity when moved to the 5' and 3' flanking regions and when inverted but is maximally active in its native intragenic site. The concerted activities of these two regulatory regions produce a 100- to 200-fold transcriptional activation during myoblast differentiation. The conserved 5' exon-intron organization of troponin I and other contractile protein genes suggests a possible mechanism by which intragenic control elements coordinate contractile protein gene regulation during skeletal myogenesis.

Complex regulation of the muscle-specific contractile protein (troponin I) gene.

A cloned quail troponin I contractile protein gene, stably transfected into a mouse myogenic cell line, exhibits appropriate developmental activation and quantitative expression during myoblast differentiation. Deletion mutagenesis analyses reveal that the troponin I gene has two distinct cis regulatory elements required for its developmental expression, as measured by mRNA accumulation and nuclear runoff transcription assays. One element in the 5' flanking region is required for maximum quantitative expression, and a second larger regulatory element (1.5 kilobases) within the first intron is responsible for differentiation-specific transcription. The upstream region is highly sensitive to negative repression by interaction with pBR322 sequences. The larger intragenic region retains some activity when moved to the 5' and 3' flanking regions and when inverted but is maximally active in its native intragenic site. The concerted activities of these two regulatory regions produce a 100- to 200-fold transcriptional activation during myoblast differentiation. The conserved 5' exon-intron organization of troponin I and other contractile protein genes suggests a possible mechanism by which intragenic control elements coordinate contractile protein gene regulation during skeletal myogenesis.

The transcription of B2 repeated sequences is regulated during the transition from quiescent to proliferative state in cultured rodent cells

The RNA polymerase III-dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus-induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus-transformed cells. In both systems, nuclear run-off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2-specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation.

Inhibition of proto-oncogene c-fos transcription by inhibitors of protein kinase C and ion transport.

Both 4 beta-phorbol 12,13-didecanoate (PDD) and calcium ionophore A23187 induced c-fos mRNA accumulation with similar kinetics in human monocyte-like cells (U937). Their effects were additive. Cells pretreated with PDD were not induced to accumulate c-fos mRNA by PDD but remained responsive to the induction by A23187. Similarly cells pretreated with A23187 responded to PDD but not to A23187. Nuclear run-off transcription assay indicated that increase in the c-fos mRNA level after the second inducer treatment corresponded to transcriptional activation of the c-fos gene. The accumulation of c-fos mRNA and the transcriptional activation of c-fos induced by PDD or A23187 were inhibited by the protein kinase inhibitor H-7 and by quinidine and amiloride. The induction by A23187, but not that by PDD, was also inhibited by 4-aminopyridine, or tetraethylammonium. From these results, it is proposed that activation of protein kinase C and Na+ or K+ transport are required for c-fos induction caused by PDD and A23187.

Transcriptional regulation of the c-myc protooncogene by 1,25-dihydroxyvitamin D3 in HL-60 promyelocytic leukemia cells.

Exposure of HL-60 promyelocytic leukemia cells to calcitriol results in a decrease in steady-state levels of c-myc mRNA and induces cellular differentiation. We have asked whether calcitriol has a direct effect on the transcription of the c-myc gene. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) decreased RNA elongation in a nuclear run-off transcription assay by 4 h after treatment. In the continuous presence of 1,25-(OH)2D3, HL-60 cell transcription of c-myc was decreased by 38% at 4 h and was abolished by 48 h. In contrast, the transcription of beta-actin was not affected by 1,25-(OH)2D3 treatment. The rate of transcription of c-myc and beta-actin was proportional to the number of nuclei and to time. Furthermore, specific hybridization of c-myc and beta-actin RNA was a linear function of RNA input. After a 48-h treatment, the c-myc/beta-actin ratio was decreased by 80-100% at [32P]RNA inputs ranging from 2 to 20 X 10(6) cpm/ml. These data temporally correlate inhibition of c-myc transcription with decreases in the steady-state levels of c-myc mRNA as assessed by Northern blot analysis. We conclude that the effect of 1,25-(OH)2D3 on c-myc expression occurs at the transcriptional level.

Insulinlike growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells.

We have previously shown that insulinlike growth factor I (IGF-I) inhibits growth hormone (GH) secretion and messenger RNA (mRNA) levels in pituitary cells. The effects of IGF-I on new GH mRNA synthesis rates in primary monolayer rat pituitary cells were therefore examined by nuclear runoff transcription assays. IGF-I (1.3 nM) treatment for 1 h inhibited GH gene transcription to 60% of controls. IGF-I (3.25 nM) maximally suppressed GH gene transcription to 30% of control values after 4 h. After 24 h treatment, GH transcription was suppressed to 48% of controls by 3.25 nM IGF-I. IGF-I (3.25 nM) also inhibited the twofold growth hormone-releasing hormone (GHRH) (10 nM)-stimulated GH gene transcription by 30% after 4 h. Transcription of the prolactin (PRL) gene was not suppressed in these cells by IGF-I. Relatively high doses of insulin (200 nM) also suppressed GH gene transcription, but epidermal growth factor and fibroblast growth factor did not change GH mRNA synthesis. The results show that IGF-I exerts a rapid and selective suppression of basal and GHRH-stimulated GH gene transcription. These data indicate a role for IGF-I in negative feedback of GH gene expression and provide evidence for the direct transcriptional regulation of the GH gene by IGF-I in primary rat anterior pituitary cells.

Transcriptional analyses of interferon-inducible mRNAs.

Using nuclear runoff transcription assays we demonstrated that alpha interferon-mediated induction of transcription of four mRNAs in HeLa monolayer cells needed ongoing protein synthesis and that such a need could be obviated by pretreating the cells with gamma interferon which, by itself, did not induce transcription of these mRNAs. In another human cell line, RD-114, synthesis of alpha interferon-inducible mRNA 561 did not need ongoing protein synthesis. In this line, however, in which interferon inhibits replication of some viruses but not of others, transcription of two of the six interferon-inducible mRNAs that we examined was not appreciably enhanced by interferon.

Transcriptional analyses of interferon-inducible mRNAs.

Using nuclear runoff transcription assays we demonstrated that alpha interferon-mediated induction of transcription of four mRNAs in HeLa monolayer cells needed ongoing protein synthesis and that such a need could be obviated by pretreating the cells with gamma interferon which, by itself, did not induce transcription of these mRNAs. In another human cell line, RD-114, synthesis of alpha interferon-inducible mRNA 561 did not need ongoing protein synthesis. In this line, however, in which interferon inhibits replication of some viruses but not of others, transcription of two of the six interferon-inducible mRNAs that we examined was not appreciably enhanced by interferon.

Varied interactions between proviruses and adjacent host chromatin.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.

Varied interactions between proviruses and adjacent host chromatin.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.

Insulin enhances transcription of the tyrosine aminotransferase gene in rat liver

The mechanism of insulin-mediated induction of tyrosine aminotransferase in rat liver was investigated using a cloned cDNA probe. The level of aminotransferase mRNA increases about fourfold following administration of the hormone. This induced mRNA accumulation does not require de novo protein synthesis. Nuclear runoff transcription assays in isolated liver nuclei demonstrate that insulin has a rapid and time-dependent stimulatory effect on aminotransferase gene transcription. The magnitude of enhanced transcription can fully account for the increase in the mRNA. We conclude that the induction of tyrosine aminotransferase in rat liver by insulin is primarily a consequence of a selective increase in the rate of transcription of the aminotransferase gene.

Tissue-specific control of rat malic enzyme activity and messenger RNA levels by a high carbohydrate diet.

In euthyroid rats fed a high carbohydrate fat-free diet for 10 days, the mass of cellular malic enzyme mRNA in liver is increased 7- to 8-fold above the basal level. Malic enzyme activity is stimulated to the same extent. This effect does not result from an increase either in the transcriptional activity of the malic enzyme gene, as determined by nuclear run-off transcription assay, or in the content of intranuclear malic enzyme RNA sequences. Mathematical modeling shows that this increase in cytoplasmic mRNA is compatible with retarded degradation of cytoplasmic mRNA. Regulation of malic enzyme by carbohydrates is liver-specific, since no response is observed in the following nonhepatic tissues: brain, heart, spleen, kidney, testis, and lung. Furthermore, the amplitude of the response in liver depends on the thyroid state of the animals, being lower (by a factor of approximately 4) in hypothyroidism and higher (12- to 15-fold) when normal animals are injected simultaneously with a daily dose of 15 micrograms of triiodothyronine per 100 g of body weight for 10 days. Since thyroid hormones regulate liver malic enzyme synthesis predominantly at the nuclear level and carbohydrates at the cytoplasmic level, the additive effect of triiodothyronine and a high carbohydrate diet on the activity of malic enzyme is readily explicable.