Early Transcriptional Regulation Research Articles

Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the adenovirus E1B oncogene.

The 19-kDa protein encoded within the adenovirus E1B gene is essential for transformation by adenovirus and for proper regulation of viral early gene transcription. In order to investigate the biological function of the 19-kDa E1B protein, vectors were constructed to produce the 19-kDa protein in mammalian cells under the direction of heterologous promoters. Surprisingly, during transient expression, the E1B 19-kDa protein specifically associated with and disrupted the organization of intermediate filaments and the nuclear lamina, without disturbing the organization of other cytoskeletal networks. These results directly demonstrate an effect of a viral transforming protein on the cytoskeleton and suggest a role for intermediate filaments and the nuclear lamina in modulation of viral gene expression and the process of oncogenic transformation.

Independent cyclic AMP and E1A induction of adenovirus early region 4 expression

A cellular transcription factor, ATF, binds to a repeated element in the adenovirus early region 4 (E4) promoter. ATF also binds to other viral early promoter regions and to the cyclic AMP (cAMP) response elements of cellular genes. In this report, we demonstrate that a single ATF-binding site located immediately upstream of the E4 TATA box, between -62 and -46, mediates induction of E4 transcription by 8-bromoadenosine-3',5-cyclic monophosphate or cholera toxin in the human hepatoma cell line HepG2 and rat pheochromocytoma cell line PC12. Different ATF-binding sites in the E4 control region independently conferred cAMP inducibility on the simian virus 40 early promoter in PC12 cells. Induction of E4 expression by cAMP was also observed in virus-infected HepG2 cells. Other viral early promoter regions that contain ATF-binding sites (E1A and E2A) were also induced by cAMP in infected cells. E4 expression was activated by the E1A 13S mRNA products in HepG2 cells. E1A trans activation appears to be distinct from the cAMP response.

Regulation of cytomegalovirus late-gene expression: differential use of three start sites in the transcriptional activation of ICP36 gene expression

We have investigated the transcriptional regulation of the human cytomegalovirus gamma gene encoding the ICP36 family (p52, the major late DNA-binding protein). The ICP36 transcription unit initiates at three distinct sites which are separated by approximately 50 nucleotides and are differentially regulated during infection. At early times (8 h postinfection), only two of these start sites, the most proximal and distal site, were active whereas at late times (36 h postinfection), the middle start site was activated. Expression from this late start site was dependent upon DNA replication. Consensus TATA elements were located upstream of all three start sites, although the element upstream of the late start site was unusual in both sequence and position when compared with conventional TATA elements. Deletion analysis was used in conjunction with transient assays to define independent promoters in this region. The two early start sites and associated TATA elements functioned as separable independently regulated promoters. The region containing the late start site and TATA element but excluding either of the flanking TATA elements was inactive in transient assays. Our work establishes that the ICP36 gene is under complex early and late transcriptional regulation and that the sequences regulating transcriptional activation are temporally and spatially distinct.

Analysis of origin of DNA replication of human papovavirus BK

We have analyzed the cis-acting sequence elements and properties of the origin of DNA replication of human papovavirus BK (BKV). The precise boundaries of the origin varied, depending on the cell type and the viral T antigen used for assay. The BKV minimal origin of replication consisted of an inverted repeat, T-antigen-binding site II, and a 20-base-pair AT block when assayed in monkey kidney CV1 and HeLa cells by using the BKV T antigen. This 76-base-pair minimal origin did not replicate in COS cells in the presence of the simian virus 40 (SV40) T antigen. Unlike that from the SV40 minimal origin, replication from the BKV minimal origin was not enhanced by BKV ori-flanking sequences in CV1 or HeLa cells, using the BKV T antigen. BKV ori-flanking sequences did activate the SV40 minimal origin of replication in COS cells and relieved the orientation-dependent property of this origin. Finally, the BKV T antigen was found to autoregulate activity of the BKV early transcriptional regulatory region. The BKV origin of replication shows similarities to and differences from those of the related viruses SV40 and polyomavirus, suggesting that the proteins involved in the initiation of replication interact with origin sequences differently in these viruses.

Mapping of two novel transcripts of bovine papillomavirus type 4.

A cDNA library was synthesized using RNA from bovine papillomavirus type 4 (BPV-4)-induced papillomas. The major viral transcript was characterized by sequencing of its cDNA, primer extension mapping and S1 nuclease protection studies. The transcript initiates at multiple sites between nucleotide (nt) 777 and nt 902, contains a splice junction between nt 1016 and nt 3376 and terminates at nt 4034, using the early polyadenylation signal at nt 4009. Sequencing of viral cDNAs and mRNA revealed deviations from the reported genomic sequence between nt 3412 and nt 3460. These resulted in a temporary frameshift, abolished the translation termination codon of the E5a open reading frame (ORF), and introduced a new termination codon in the E4 ORF. The new uninterrupted E5 ORF was found to have greater homology to the E4 ORFs of other papillomaviruses than to their E5 ORFs. The E5 ORF of BPV-4 is joined to a five codon ORF in the leader exon of the major transcript, in the same way as the E4 ORF in the major transcripts of BPV-1 and human papillomavirus type 11. On this basis, it has been redesignated E4, and the viral genomic sequence revised. Two novel transcriptional promoters of BPV-4 were defined: a putative controller of the multiple major RNA start sites around nt 870 and a minor TATA box at nt 691. In addition, minor early region transcripts were mapped which initiate between nt 3071 and nt 3152. None of these transcripts utilizes the splice site at nt 3376. These messages may express E2-encoded functions.

Sequence and genetic organization of adenovirus type 35 early region 3.

The early transcription region 3 (E3) of group B adenovirus type 35 (Ad35), a serotype isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders, has been partially sequenced. We had previously identified an Ad35 29-kilodalton (kDa) early glycoprotein which, analogous to group C Ad2 E3-19K, associated with major histocompatibility complex class I antigens in the endoplasmic reticulum of infected cells. The open reading frame (ORF) of the Ad35 29-kDa protein has now been identified within a 2-kilobase-pair cloned Ad35 E3 fragment. The predicted amino acid sequence was very similar to that of group B Ad3 E3-19K. In contrast, homology between the Ad35 and Ad2 glycoproteins was limited to five cysteines in identical positions and a 20-amino-acid region proximal to the transmembrane domain. In addition, 20.3- and 20.6-kDa ORFs have been identified downstream from the ORF for the Ad35 glycoprotein. Analogous 20-kDa ORFs are present in the Ad3 E3 region but are not present in Ad2 and Ad5. In contrast, the region analogous to an Ad2 11.6-kDa ORF, which is 9 kDa in size in Ad3, was absent from the expected position within the Ad35 E3 region. Because the E3 region is likely to play an important role in the interaction between virus and host, analysis of the function of the Ad35 E3 proteins should further our understanding of adenovirus pathogenesis.

Cellular transcription factor binds to adenovirus early region promoters and to a cyclic AMP response element.

We have analyzed the sequences that regulate the transcription of adenovirus type 5 early region 4 (E4). A region located immediately upstream of the E4 TATA box is required for efficient E4 transcription in vitro and in vivo. A cellular transcription factor, termed ETF-A, binds to this region. ETF-A also binds to additional sites in the E4 5'-flanking region, including the adenovirus terminal repeat, as well as to the adenovirus early region 2 promoter region and the adeno-associated virus early promoter region. A repeated sequence motif is found in each binding domain. The same factor binds to a region upstream of a cellular gene that contains a cyclic AMP response element. Consistent with this result, E4 expression is induced in vivo by cyclic AMP. Two other regions further upstream of the E4 initiation site also contribute to efficient E4 expression. These domains are functionally redundant and contain binding sites for ETF-A. One domain is the adenovirus terminal repeat, which has strong promoter activity in vitro and in vivo.

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4.

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4.

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

Topoisomerase I interaction with SV40 DNA in the presence and absence of camptothecin.

Camptothecin is an antitumor drug, which is a specific inhibitor of eukaryotic topoisomerase I. Enzyme inhibition is related to the stabilization of cleavable complexes between topoisomerase I and DNA. The genomic and DNA sequence localization of L1210 topoisomerase I-mediated DNA breaks produced by camptothecin were determined in the SV40 genome. DNA cleavage was predominantly single-stranded and localized in selective regions of the DNA. A major cleavage site was found at nucleotide 4995 on the coding strand in the early transcription region. The DNA sequence was determined at prominent cleavage sites (nucleotides 127 and 199 in the two 72 bp repeats and nucleotide 4955). A DNA consensus sequence 5'-GATG-3' was found in SV40 DNA. Cleavage occurred between the T and the G and topoisomerase I was linked to the 3'-DNA terminus at the T position. The sequence GATG is more frequent in the non transcribed strand of the early and late transcription of SV40 than in the transcribed strands. This finding is consistent with the role of topoisomerase I in transcription.

Detailed kinetics of adenovirus type-5 steady-state transcripts during early infection

The appearance and steady-state accumulation of specific viral RNAs during the early phase of adenovirus type 5 (Ad5) infection was examined. HeLa cells were synchronously infected and harvested at 30 min intervals throughout the first 12 h of infection. Total cytoplasmic RNA was extracted from infected cells and analyzed by hybridization-selection and translation to identify the viral mRNAs from each early region on the basis of the protein products they encode. The same RNA samples were used for S-1 nuclease and Northern blot analyses to quantitatively compare the levels of individual viral RNAs that accumulate within each early transcription region (E1A, E1B, LI, E2A, E3 and E4). The salient features of this analysis show that RNA accumulation occurs first from E1A followed by E2A, E3 and E4, E1B and lastly, LI. Although the profile of RNA accumulation was unique for each early region, overlapping RNAs within E1A, E3, and E4, respectively, remained generally parallel to one another throughout early infection, in contrast to RNAs from E1B and LI, respectively. Since both the appearance and quantitative accumulation of specific early viral mRNAs were examined at many time points, a number of subtleties associated with the complex dynamics of early Ad5 gene expression were revealed. In particular, the L1 region was shown to transcribe from the major late promoter two early RNAs of 3.81 Kb and 3.5 Kb, either or both of which encode the 52,55 kDa proteins; the auxiliary i leader sequence was found on the 3.81 Kb RNA but not on the 3.5 Kb RNA.

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons.

Regulation of in vitro and in vivo transcription of early-region IV of adenovirus type 5 by multiple cis-acting elements.

A series of deletion mutants spanning the promoter of the adenovirus early-region IV (EIV) gene were tested for transcriptional activity, using both in vitro and in vivo assays. Four distinct domains had additive effects on efficient transcription from the EIV promoter in HeLa whole-cell extracts. The first resided 20 to 27 bases upstream of the initiation site and included the TATA box. Deletion of the TATA box drastically reduced the transcriptional activity in vitro but had a lesser effect in vivo. The second region extended from -32 to -177 and contained two 17-base-pair inverted repeats, centered around -40 and -162. Sequences lying between -140 and -173 were important for efficient transcription since deletion of this region reduced the activity fourfold. Deletion of either one of the two inverted repeats or insertion of DNA fragments between them resulted in the synthesis of extra transcripts that initiated at sites upstream from the EIV site. The third region was located between -198 and -250 and contains three guanosine-plus-cytosine-rich sequences, present around -212 (GGGCGG), -233 (GGGCGG), and -251 (CGCGGG). The fourth, most upstream region was located between -260 and -307. Deletion of this region, which contains the NF-1 factor-binding site, slightly reduced transcriptional activity both in vivo and in vitro. The data indicate that multiple cis-acting elements are required for efficient transcription from the EIV promoter in both in vitro and in vivo systems.

Regulation of in vitro and in vivo transcription of early-region IV of adenovirus type 5 by multiple cis-acting elements.

A series of deletion mutants spanning the promoter of the adenovirus early-region IV (EIV) gene were tested for transcriptional activity, using both in vitro and in vivo assays. Four distinct domains had additive effects on efficient transcription from the EIV promoter in HeLa whole-cell extracts. The first resided 20 to 27 bases upstream of the initiation site and included the TATA box. Deletion of the TATA box drastically reduced the transcriptional activity in vitro but had a lesser effect in vivo. The second region extended from -32 to -177 and contained two 17-base-pair inverted repeats, centered around -40 and -162. Sequences lying between -140 and -173 were important for efficient transcription since deletion of this region reduced the activity fourfold. Deletion of either one of the two inverted repeats or insertion of DNA fragments between them resulted in the synthesis of extra transcripts that initiated at sites upstream from the EIV site. The third region was located between -198 and -250 and contains three guanosine-plus-cytosine-rich sequences, present around -212 (GGGCGG), -233 (GGGCGG), and -251 (CGCGGG). The fourth, most upstream region was located between -260 and -307. Deletion of this region, which contains the NF-1 factor-binding site, slightly reduced transcriptional activity both in vivo and in vitro. The data indicate that multiple cis-acting elements are required for efficient transcription from the EIV promoter in both in vitro and in vivo systems.

Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.

A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium from transfected cells inhibits binding of 125I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.

Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus.

Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789-4793, 1983). This observation was based on our inability to propagate an adenovirus type 5 mutant lacking functional VAI and VAII genes. Subsequently, it was found that this mutant was viable and able to grow to a low titer. Therefore, we examined the complementation of the VAI RNA function by the Epstein-Barr virus-encoded RNAs by constructing additional adenovirus type 5 substitution mutants containing multiple copies of the Epstein-Barr virus-encoded RNA genes in nonessential early transcriptional region III. The new substitution mutants synthesized viral polypeptides at late times at levels comparable to those observed in wild type-infected cells. Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells.

The expression of the SV40 early region in the transformed human cell line SV80

The one complete copy of the SV40 early region present in human cells of the transformed line SV80 carries a duplication of the 5' portion of the early region, including its transcriptional control region and splicing signals (W. Gish and M. Botchan, personal communication). Novel SV40-specific RNA species of sufficiently large size, 3.8 and 4.2 kb, to be expressed from the duplicated early transcriptional control region were detected in SV80 cytoplasmic and nuclear RNA preparations by blot hybridization. The results of transcription in a cell-free system of a plasmid, pSV80-04, representing this SV80 cell SV40 DNA integrate (W. Gish and M. Botchan, personal communication) and of nuclease protection experiments with end-labelled pSV80-04 DNA fragments support the conclusion that the duplicated early sequences are transcribed in SV80 cells. It has also been established that the duplicated early splicing signals are functional in SV80 cells. These results are discussed in relation to the large amounts of SV40 early mRNA and T-antigen synthesized in cells of the SV80 line.

Unusual regulation of simian virus 40 early-region transcription in genomes containing two origins of DNA replication.

As part of our efforts to create multifunctional vectors for the transduction of animal cells, a set of simian virus 40 recombinants were constructed which contain an inverted duplication of the region including the origin of viral DNA replication (ori) and the early-region promoter. The unusual aspects of the structure of these recombinant genomes revealed several unexpected features of their function. In particular, transcription from the early-region promoters on these recombinants occurred primarily after the start of DNA replication, and, in that sense, these promoters behaved as if they were late-region promoters. This behavior results from the fact that these genomes contain multiple ori segments, and, therefore, they replicate earlier and faster than wild-type virus DNA, thereby causing a precocious shift in the initiation of early-region transcription from sites downstream of ori to sites located upstream of ori. The abnormal expression from multiple ori genomes is consistent with our present notions regarding the replication-dependent shift in early-region transcriptional start sites (Buchman et al., Mol. Cell. Biol. 4:1900-1914). Since our experiments demonstrate that RNAs initiated upstream of ori contribute to T-antigen formation late in infection, we suggest that the shift in early-region transcription starts modulates large T-antigen production in concert with viral DNA replication.

Unusual regulation of simian virus 40 early-region transcription in genomes containing two origins of DNA replication.

As part of our efforts to create multifunctional vectors for the transduction of animal cells, a set of simian virus 40 recombinants were constructed which contain an inverted duplication of the region including the origin of viral DNA replication (ori) and the early-region promoter. The unusual aspects of the structure of these recombinant genomes revealed several unexpected features of their function. In particular, transcription from the early-region promoters on these recombinants occurred primarily after the start of DNA replication, and, in that sense, these promoters behaved as if they were late-region promoters. This behavior results from the fact that these genomes contain multiple ori segments, and, therefore, they replicate earlier and faster than wild-type virus DNA, thereby causing a precocious shift in the initiation of early-region transcription from sites downstream of ori to sites located upstream of ori. The abnormal expression from multiple ori genomes is consistent with our present notions regarding the replication-dependent shift in early-region transcriptional start sites (Buchman et al., Mol. Cell. Biol. 4:1900-1914). Since our experiments demonstrate that RNAs initiated upstream of ori contribute to T-antigen formation late in infection, we suggest that the shift in early-region transcription starts modulates large T-antigen production in concert with viral DNA replication.

Complex regulation of simian virus 40 early-region transcription from different overlapping promoters.

During simian virus 40 lytic infection there is a shift in initiation sites used to transcribe the early region, which encodes large T and small t antigens. Early in infection, transcription is initiated almost exclusively from sites that are downstream of the origin of DNA replication, whereas transcripts produced later are initiated mainly from sites on the upstream side. We have used mutant virus and specially constructed plasmid DNAs to investigate the factors regulating this transcriptional shift. In our studies simian virus 40 large T antigen appears to mediate the shift in transcription in two ways: first, T antigen represses transcription at the downstream sites late in infection by binding to the region where these RNAs are initiated; second, T antigen promotes transcription from sites on the upstream side by its ability to initiate replication or amplification, or both, of the template DNA. In addition, transcription from the downstream sites is heavily dependent on enhancer sequences located in the 72-base-pair repeat region, whereas transcription from the upstream sites late in infection does not require enhancer sequences. Thus, different overlapping promoters regulate simian virus 40 early-region expression in a manner that apparently coordinates the production of large T antigen with the increase in viral DNA.