Tk Promoter Research Articles

The Evi-1 proto-oncogene encodes a transcriptional repressor activity associated with transformation.

The myeloid transforming gene Evi-1 encodes a protein with two zinc finger domains, designated ZF1 and ZF2, with distinct DNA binding specificities. For the first time we demonstrate that Evi-1 has transcriptional repressor activity which is directly proportional to the amount of Evi-1 protein in cells. Repression has been observed with two distinct promoters: the minimal HSV-1 tk promoter and a VP16 inducible adenovirus E1b minimal promoter. Optimal repression is DNA binding dependent and is mediated by either ZF1 or a heterologous GAL4 DNA binding domain (GAL4DBD) but is significantly less efficient through the ZF2 binding site. Both GAL4DBD/Evi-1 fusion and non-fusion proteins have been used to map the repressor activity to a proline-rich region located within amino acids 514-724 between the ZF1 and ZF2 domains. Constitutive expression of mutant proteins lacking the repressor domain are defective for transformation of Rat1 fibroblasts demonstrating that this region is required for the oncogenic activity of the Evi-1 protein. These studies show that the Evi-1 gene encodes a transcriptional repressor and has important implications for the mechanism of action of the Evi-1 protein both in development and in the progression of some myeloid leukaemias.

Inhibition of Estrogen-Induced Activity by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-7 Human Breast Cancer and Other Cell Lines Transfected with Vitellogenin A2 Gene Promoter Constructs1

The antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in several cell lines using transient transfection assays and constructs containing 5′-regulatory sequences from the estrogen (E2)-responsive vitellogenin (Vit) A2 gene linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. TCDD significantly inhibited CAT activity induced by E2 in MCF-7 human breast cancer cells transiently transfected with 5′-deletion plasmids containing the homologous promoter [(−821/+14)- and (−482/+14)-CAT] or the heterologous thymidine kinase (tk) promoter [(−821/−87)tk-, (−482/−87)tk-, (−397/−87)tk-, and (−331/−87)tk-CAT]. In parallel experiments using wild-type mouse Hepa 1c1c7 and human HeLa cells cotransfected with a human estrogen receptor expression plasmid, TCDD also inhibited E2-induced CAT activity. The role of the nuclear Ah receptor complex was confirmed by results of the following studies using MCF-7 or mouse Hepa 1c1c7 cells transiently transfected with E2-responsive Vit A2 gene 5′-promoter constructs: (i) for a series of Ah receptor ligands, there was a correlation between their antiestrogenic activity in MCF-7 cells and their rank order binding affinity for the Ah receptor; (ii) α-naphthoflavone, an Ah receptor antagonist, inhibited the antiestrogenic activity of TCDD in MCF-7 cells; and (iii) TCDD inhibited E2-induced CAT activity in Ah-responsive wild-type but not in Ah-nonresponsive class 2 mutant Hepa 1c1c7 cells. The antiestrogenic activity of TCDD was also observed in cells which transiently overexpressed the human estrogen receptor (ER), suggesting that the mechanism does not involve downregulation of the ER by TCDD.

Effects of an internal transcription unit and its orientation on retrovirus titre and expression.

Using the retroviral vector pMsp, constructs were produced with different coding sequences under the control of the Herpes simplex virus type 1 (HSV) thymidine kinase (tk) promoter, with the internal coding sequence in the same or reverse orientations with respect to long terminal repeat (LTR)-driven transcription and with or without an internal tk polyadenylation (polyA) signal. Following introduction of these constructs into ecotropic or amphotropic packaging cell lines by transfection or infection, it was found that, most consistently, those constructs in which the internal coding sequence/polyA signal component was inserted in the same orientation as LTR-driven transcription produced lower titres of virus than those in which this component was inserted in the reverse orientation. Also, in a construct containing a coding sequence in the same orientation as LTR-driven transcription, but lacking an internal polyA signal, virus titre was much greater than in the corresponding construct possessing the internal polyA signal. Additionally, although functional assays have previously demonstrated expression of the inserted sequences, transcription from the internal tk promoter was inefficient in all these constructs.

Enhancers of hypermutation.

Hypermutation at the immunoglobulin (Ig) loci increases the mutation rate more than 10(5)-fold over the normal, spontaneous rate. We studied two kinds of cis-acting elements - 3' enhancers and promoters - in a system in which a gene encoding the mu heavy (H) chain (Igm) is transfected in vitro into a cell line with an active Ig mutator. A construct containing a rearranged Igm gene requires the 3' H enhancer for hypermutation at a rate comparable with the one at the endogenous gene segment encoding the H chain variable region (V). Without the 3' enhancer, the basal mutational activity is much lower, but still higher than the normal, spontaneous mutation rate. Replacement of the 3' H enhancer by atopic elements of similar function also supports full hypermutation. Even though these 3' elements are defined as transcriptional enhancers, they do not seem to increase hypermutation via an increase in the rate of transcription. Replacement of the endogenous promoter by the tk promoter slightly increases hypermutability of the construct; thus, no specific sequences in the Ig promoter are likely to target hypermutation.

Estrogen and thyroid hormone interaction on regulation of gene expression.

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.

Human insulin gene expression in transgenic mice: mutational analysis of the regulatory region

A mini-human insulin gene and four derivatives mutated at several regions potentially involved in the regulation of gene expression were used to generate transgenic mouse lines. The effect of these mutations on the efficiency of gene expression and cell specificity was studied using three approaches: (1) Northern blot analysis using total RNA from pancreas and other organs, (2) radioimmunoassay to detect the human C-peptide in urine samples, and (3) immunocytochemistry of pancreas sections to examine whether expression of the transgene was still specifically expressed in β-cells. Mutation of the cis-acting elements located between −238 and −206 (GCII and CTII motifs) resulted in a strong decrease of gene expression in the pancreas of transgenic mice, but it did not lead to complete extinction of the transgene expression. This region alone (−255/−202), when linked to the minimal Herpes simplex virus thymidine kinase gene (tk) promoter, failed to activate chloramphenicol acetyltransferase (CAT) gene expression in transfected insulinoma cells, while it was activated by the equivalent region of the rat insulin I gene. On the contrary, mutation of the DNA motifs located between −109 and −75 (GCI and CTI) or between −323 and −297 (CTIII) did not significantly affect the level of the human insulin gene expression in transgenic mice. Replacement of the insulin promoter (−58/+1) by the tk promoter did not alter its level of expression in transgenic mice. In all instances, expression of the different transgenes remained localized in the islet β-cells. Altogether, these results indicate that the GCII-CTII motif is an important regulatory element for efficient expression of the human insulin gene in vivo, although it alone does not allow gene expression as it would require the association of other elements.

Differential Regulation of c-Myb-induced Transcription Activation by a Phosphorylation Site in the Negative Regulatory Domain

The c-myb protooncogene encodes a highly conserved 75-89-kDa transcription factor that contains three functional domains, an amino-terminal DNA binding domain (DBD), a central acidic transactivation domain, and a carboxyl-terminal negative regulatory domain (NRD). Two acute transforming retroviruses, avian myeloblastosis virus and the E26 leukemia virus, transduced portions of c-myb and encode Myb proteins that are truncated in both the DBD and the NRD. Several conserved potential sites for phosphorylation by proline-directed serine/threonine protein kinases reside in or near the NRD, suggesting that phosphorylation might play a role in regulating c-Myb. We have previously demonstrated that serine 528, located in the NRD, is a target for p42(mapk) in vitro. Serine 528 is phosphorylated in vivo in several cell lines, and substitution of serine 528 to alanine (S528A) resulted in an increased ability of Myb to transactivate a synthetic promoter containing five copies of the mim-1A Myb-responsive element and a minimal herpes tk promoter. We have tested the ability of S528A Myb to transactivate a series of cellular target promoters and report that the serine to alanine substitution increased the ability of Myb to activate transcription from the CD34 promoter but not the c-myc or mim-1 promoters. This suggests that phosphorylation of serine 528 may differentially regulate c-Myb activity at different promoters. The DNA binding and multimerization activities of c-Myb appear to be unaffected by the S528A substitution, suggesting that phosphorylation of serine 528 may mediate its effect on the transcription transactivating activity of c-Myb by regulating interactions with other proteins.

Identification of the human insulin negative regulatory element as a negative glucocorticoid response element

Insulin gene transcription in adults is restricted to pancreatic beta cells. Studies with both transgenic mice and islet cell lines have demonstrated that beta cell specific expression is conferred by the 5′ flanking region of the insulin gene. Transfection analysis has shown that cell specific expression involved an interaction between both positive and negative promoter cis elements. An upstream region (between −258 and −279) of the human insulin promoter served as a site of negative regulation. Transfection analysis in the pancreatic cell line HIT T-15 M 2.2.2 revealed that a DNA fragment containing this region causes a 45% reduction in promoter activity when linked to the native insulin promoter and a 72% reduction when linked to a heterologous tk promoter. Electrophoretic mobility shift analysis of this negative regulatory region (NRE) reveals a complex pattern of binding, wherein two major and several minor complexes are observed. Competition experiments demonstrated that formation of the fastest mobility complex is completely inhibited with excess cold glucocorticoid responsive element (GRE) consensus oligonucleotide. Purified glucocorticoid receptor binding domain (T7X556) demonstrated binding to the NRE oligonucleotide. Functional studies showed that dexamethasone treatment of HIT T-15 M 2.2.2 cells containing an NRE-tk CAT plasmid decreased CAT gene expression by 48%. Analysis of the NRE revealed 73% homology with the negative GRE consensus sequence. These data show that the human insulin NRE is a negative glucocorticoid response element.

Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

Use of Epstein-Barr virus nuclear antigen-1 in targeted therapy of EBV-associated neoplasia.

To target expression of toxic genes to Epstein-Barr virus (EBV)-associated tumor cells, we have developed an EBV-driven enzyme prodrug system (EDEPS) that takes advantage of the trans-activating properties of EBNA1, a latent protein expressed in all EBV-containing cells, to direct expression of cytosine deaminase (CD) at high levels in those cells only. Plasmids were constructed in which the CD gene or a luciferase reporter gene were cloned downstream of the herpes simplex virus thymidine kinase (tk) promoter and the family of repeats (FR) sequence from the oriP region of EBV. Analysis of luciferase activity after transient transfection into a panel of EBV-negative or -positive human cell lines showed that the presence of the FR element enhanced transcription from the tk promoter in all EBV-positive cell lines, whereas transcription from tk was repressed in all EBV-negative cell lines, including B, T, and fibroblast cell lines. In clonogenicity assays following transfection with the CD vector, the presence of 5-fluorocytosine (5-FC) in the culture medium completely abolished cell growth in EBV-positive cell lines, but did not affect the growth of EBV-negative cell lines. This vector system should have wide applicability in that it allows targeted expression of any gene of interest to tumors that carry EBV, irrespective of the role EBV plays in their pathogenesis.

Trapping of mammalian promoters by Cre-lox site-specific recombination.

One of the challenges in human genome research is to identify the promoter sequences which play a key role in the regulation of gene expression. We report here a new promoter trapping system for use with mammalian cells comprised of the following three steps: 1) Cloning of DNA fragments into a promotertrapping vector, 2) integration of the trapping vector into a designated target in the mammalian genome using the Cre site-specific recombinase, and 3) screening of integrants for trapped promoter sequences by activation of the luciferase gene. To assess the efficiency of this system, lox trapping vectors containing sense tk promoter, antisense tk promoter, or a non-promoter sequence of the neo gene were employed. The resulting levels of luciferase activity of the site-specific integrants were measured directly. Luciferase activity of the integrants can be assayed under conventional culture conditions by simply replacing the culture medium with potassium phosphate buffer containing luciferin. Only those G418r colonies carrying the tk promoter in the normal orientation exhibited a 21-to 35-fold increase in luciferase activity over that of the other integrants. These results indicate that this system is an effective means of trapping promoter sequences from random mammalian genomic DNA fragments.

Identification of a negative regulatory element in the 5'-flanking region of the human dopamine beta-hydroxylase gene.

Transient transfection experiments indicate that a 5'-flanking upstream domain, residing between -437 and -262 bp of the human dopamine beta-hydroxylase (DBH) gene, has a cell type-specific silencer function. This domain contains a putative silencer motif (which we designate DBH negative regulatory element, DNRE), showing sequence homology with the neural-restrictive silencer element (NRSE or RE-1) recently characterized in type II sodium channel, SCG10 and synapsin I genes. When the DNRE was placed at the proximal 262 bp of the homologous (DBH) promoter, it exhibited strong silencer activity both in DBH-expressing SK-N-BE(2)C as well as in DBH-nonexpressing HeLa cells. In addition, the DNRE also exhibited modest silencer activity upon a heterologous tk (herpes simplex virus thymidine kinase) promoter in both cell lines. Electrophoretic mobility shift assay demonstrated that nuclear extracts from both SK-N-BE(2)C and HeLa cells contain protein(s) that specifically bind to the DNRE. Formation of this DNRE/protein complex was specifically inhibited by an excess of unlabeled DNRE or NRSE. Finally, a similar sequence motif residing in the corresponding upstream area of the rat DBH gene also had a negative regulatory function, indicating that the silencer function of the DNRE is conserved in human and rat DBH genes.

Effect of Expression of the Aphthovirus Protease 3C on Viral Infection and Gene Expression

Cells transformed with specific regions of the foot-and-mouth disease virus (FMDV) genome have been constructed and analyzed with respect to viability and susceptibility to FMDV infection. Constitutive expression of an active protease 3C under the control of the tk promoter has been documented by the ability of transformed cells to catalyze the processing of a P1 capsid precursor. High-level, transient expression but not low-level, constitutive expression, of 3C caused a 10-fold reduction in the yield of FMDV and was detrimental to the expression of the cotransfected reporter luciferase gene. No such effect was observed in assays involving cells transfected with a deleted, inactive form of 3C. The negative effect of 3C was not observed when the same reporter gene was integrated and expressed in a constitutive fashion nor when its translation was directed by the internal ribosome entry site element of FMDV in transient expression assays. The results show that cells with a low level of expression of the aphthoviral 3C can be stably maintained and can provide a useful tool to study polyprotein processing.

Differential use of the regulatory elements of the αB-crystallin enhancer in cultured murine lung (MLg), lens (αTN4-1) and muscle (C2C12) cells

The mouse αB-crystallin-encoding gene ( αB-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated αB-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg αB-cry transcripts comigrated with αB-cry transcripts contained in total and poly(A) +RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both αB-cry promoter/ cat reporter gene constructs and αB-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter ( ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from αTN4-1 lens and C2C12 muscle cells. The αB-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in αTN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, αBE-1 (nt -407 to -397), αBE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only αBE-1 and αBE-2. Previous data have shown that lens cells use αBE-1, αBE-2 and αBE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.

The Retinoblastoma Susceptibility Gene Product Represses Transcription When Directly Bound to the Promoter

Rb represses E2F-mediated transcription in part by blocking the trans-activation domain of E2F. In addition, Rb can convert an E2F binding site from a positive to a negative element. To examine the effect of a Rb-DNA-bound complex on transcription, full-length Rb was fused to the DNA binding domain of GAL4. Here, we report that GAL4-Rb can repress transcription mediated by either Sp1, AP-1, or p53, dependent upon the presence of both the GAL4 DNA binding domain and GAL4 binding sites. Moreover, GAL4-Rb inhibited the activity of the herpes simplex virus tk promoter from GAL4 binding sites located at a distance from the promoter. In contrast, GAL4-Rb was unable to repress basal transcription. Cotransfection of specific cyclins and cyclin-dependent kinases or SV40 T-antigen abolished the repressive activity of GAL4-Rb. The domains of Rb involved in mediating the repression of transcription were mapped to regions that are overlapping, but not identical, to those required for the interaction with E2F. We propose that Rb can function as a general repressor of transcription when bound to the promoter region.

Protein that binds to the distal, but not to the proximal, CCAAT of the human thymidine kinase gene promoter

Mobility shift assays were used to examine protein binding to the human TK gene CCAAT boxes. Similar protein binding patterns were observed with probes containing either the proximal or distal CCAAT. However, probes containing both CCAAT boxes in which one of the CCAAT boxes was inactivated by mutation did not demonstrate identical binding patterns. One of the complexes formed with the longer probes was only observed when the distal CCAAT was intact. This species was not formed with probes that only contained an intact proximal CCAAT, and its formation could only be competed by oligonucleotides containing the distal CCAAT motif. This observation reveals the existence of a protein that can bind to the distal, but not to the proximal, CCAAT of the human TK promoter. This protein may account for the previous observation that the two CCAAT motifs are not functionally equivalent. The protein that binds to the distal, but not to the proximal, CCAAT (DTK-CBP) was also present in two human cell lines. Significantly more DTK-CBP was present in nuclear extracts of HepG2 and WI38 cells than in TK-ts13 cells. However, this protein was not observed in three different murine cell lines and one primary culture. Its abundance in some human cell lines suggests it might modulate the expression of human TK mRNA in cells that express this protein.

996-12 Overexpression of Sarcoplasmic Reticulum Calcium ATPase in Neonatal Cardiomyocytes Using a Recombinant Adenovirus

Cardiac Sarcoplasmic Reticulum Calcium ATPase (SERCA2) is a membrane pump that plays a major role in regulating calcium levels in cardiac myocytes. Recent data have documented a decrease in SERCA2 levels in human heart failure. In order to further study the importance of SERCA2 levels in cardiac cellular physiology we constructed a recombinant replication deficient adenovirus encoding SERCA2 driven by an SV40 enhanced TK promoter (14CTK). After documenting the presence of the SERCA2 gene by southern analysis of viral DNA. neonatal rat cardiomyocytes were infected with either the SERCA2 encoding adenovirus or an adenovirus encoding beta-galactosidase. Cells were harvested for RNA preparation at 48 hours and for protein at 72 hours post infection. Northern blot analysis using a full length probe specific for rat SERCA2 revealed a several fold induction of SERCA2 mRNA levels compared to cells infected with the beta-galactosidase encoding virus. Western blot analysis with a polyclonal SERCA2 antibody revealed an approximate 2.5 fold induction of SERCA2 protein. These data demonstrate that SERCA2 can be overexpressed in cardiac myocytes using this adenoviral vector based approach. Functional studies of infected cardiomyocytes are currently underway.

Characterization of the human glucocorticoid receptor promoter.

To elucidate the functional elements that are involved in the regulation of the human glucocorticoid receptor (hGR) gene, transient expression, DNase I footprinting, and gel mobility shift analyses were conducted. We found that the hGR promoter region between -700 and +38 bp contained 11 footprinted sites. Deletion of the -374 to -183 bp region, which is highly conserved between human and mouse (93%), induced a 5-24-fold reduction in promoter activity in HeLa, NIH3T3, CV1, and HepG2 cells. Three footprints, FP5, FP6, and FP7, were shown to map to this region. In particular, the FP7 site was found to be within the -374 to -347 bp region. Deletion of this region triggered a significant decline in promoter activity in HeLa and NIH3T3 cells but not in HepG2 cells. AP2 was found to bind FP7. In HepG2 cells AP2 elicited transactivation of the hGR promoter activity. Transfection data revealed that the upstream GC box-rich fragment between -700 and -375 bp induced a 4-7-fold activation of the heterologous tk promoter in an orientation-independent manner. Our studies demonstrate that several transcription factors are involved in regulating GR expression and that AP2 could function as an important positive regulator of GR promoter activity.

Nuclear degradation of nonsense mutated beta-globin mRNA: a post-transcriptional mechanism to protect heterozygotes from severe clinical manifestations of beta-thalassemia?

Nonsense mutations of the beta-globin gene are a common cause of beta-thalassemia. It is a hallmark of these mutations not only to cause a lack of protein synthesis but also a reduction of mRNA expression. Both the pathophysiologic significance and the underlying mechanisms for this surprising phenomenon have so far remained enigmatic. We report that the reduction of the fully spliced mutant beta-globin mRNA already manifests itself within the nucleus. In contrast, the levels of mutant pre-mRNA are normal. The promoter and the 5'-untranslated region (5'-UTR) of the herpes simplex virus type 1 thymidine kinase (HSV1 Tk) gene can independently circumvent this recognition/response mechanism in cis and restore nonsense mutated beta-globin mRNA expression to normal levels. These two genetic elements can thus exert a dominant influence on the post-transcriptional control of nonsense mutated beta-globin gene expression. While wild-type mRNA levels are restored by fusion of the HSV1 Tk 5'-UTR to the nonsense mutated beta-globin reading frame, translation of a wildtype reading frame in such a hybrid is precluded. In contrast, the HSV1 Tk promoter appears to efficiently deliver the mRNA to the translational apparatus. The 5'-UTR and the promoter sequences therefore control the nuclear fate of nonsense mutated beta-globin mRNA by separable pathways. The nuclear mRNA degradation mechanisms examined here may prevent the synthesis of C-terminally truncated beta-globin chain fragments and may protect heterozygotes from clinically relevant symptoms of beta-thalassemia.

Requirements for activation of the herpes simplex virus glycoprotein C promoter in vitro by the viral regulatory protein ICP4

During infection with herpes simplex virus, infected-cell polypeptide 4 (ICP4) activates transcription of most herpes simplex virus genes. In the present study, the mechanism of activation of transcription by ICP4 was investigated by using a reconstituted in vitro system with fractionated and purified general transcription factors, coupled with DNA-binding assays. The templates used in the reactions included regions of the gC and thymidine kinase (tk) promoters in plasmids, and on isolated fragments, allowing for the evaluation of the potential function of naturally occurring and inserted ICP4-binding sites and elements of the core promoter. ICP4 efficiently activated transcription of the gC promoter by facilitating the formation of transcription initiation complexes. ICP4 could not substitute for any of the basal transcription factors. Moreover, TATA-binding protein (TBP) could not substitute for TFIID in activation, suggesting a requirement for TBP-associated factors. Interactions between ICP4 and DNA 3' to the start site was necessary for activation of the gC promoter. The requirement for DNA-protein contacts could be met either by the presence of an ICP4-binding site in the gC leader, by the presence of a site more than 150 nucleotides further downstream, by an inserted site that normally acts to repress transcription, or by the addition of sufficient non-site-containing DNA. The gC TATA box and start site, or initiator element (inr), were individually sufficient for activation by ICP4 and together contributed to optimal activation. In contrast to gC, the tk promoter was poorly activated in the reconstituted system. However, the tk TATA box was efficiently activated when the tk start site region was replaced with the gC inr, suggesting that activation was mediated through the inr and inr-binding proteins. In addition, mutation of the inr core resulted in a gC promoter that was very poorly activated by ICP4. The results of this and previous studies demonstrate that ICP4 activates transcription in a complex manner involving contacts with DNA 3' to the start site, TBP, TFIIB, TBP-associated factors, and possibly proteins functioning at the start site of transcription.