Major Late Promoter Research Articles

Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization

Subspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism.

Hyaluronidase Expression by an Oncolytic Adenovirus Enhances Its Intratumoral Spread and Suppresses Tumor Growth

Successful virotherapy requires efficient virus spread within tumors. We tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix (ECM), could enhance the intratumoral distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in mice-bearing tumor xenografts improves the antitumor activity of an oncolytic adenovirus. Next, we constructed a replication-competent adenovirus expressing a soluble form of the human sperm hyaluronidase (PH20) under the control of the major late promoter (MLP) (AdwtRGD-PH20). Intratumoral treatment of human melanoma xenografts with AdwtRGD-PH20 resulted in degradation of hyaluronan (HA), enhanced viral distribution, and induced tumor regression in all treated tumors. Finally, the PH20 cDNA was inserted in an oncolytic adenovirus that selectively kills pRb pathway-defective tumor cells. The antitumoral activity of the novel oncolytic adenovirus expressing PH20 (ICOVIR17) was compared to that of the parental virus ICOVIR15. ICOVIR17 showed more antitumor efficacy following intratumoral and systemic administration in mice with prestablished tumors, along with an improved spread of the virus within the tumor. Importantly, a single intravenous dose of ICOVIR17 induced tumor regression in 60% of treated tumors. These results indicate that ICOVIR17 is a promising candidate for clinical testing.

Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site

The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Two proteins from the adenoviral L4 unit have been suggested as DEF-A candidates. Here we have examined L4-22K and L4-33K for possible DEF-A activity. We show that L4-22K stimulates transcription from the MLP in a DE sequence dependent manner both in vivo and in vitro, and that L4-22K binds to the DE sequence in vitro. Further, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.

Adenovirus Late-Phase Infection Is Controlled by a Novel L4 Promoter

During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.

80. Replicating Adenoviruses That Harbors the ADP Protein and the Sodium Iodide Symporter Protein (NIS) under the Control of the Major Late Adenovirus Promoter

80. Replicating Adenoviruses That Harbors the ADP Protein and the Sodium Iodide Symporter Protein (NIS) under the Control of the Major Late Adenovirus Promoter

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

TAF4/4b·TAF12 Displays a Unique Mode of DNA Binding and Is Required for Core Promoter Function of a Subset of Genes

The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b.TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is approximately 70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes.

482. Replicating Adenoviruses That Harbors the ADP Protein and the Sodium Iodide Symporter Protein (NIS) under the Control of the Major Late Adenovirus Promoter

482. Replicating Adenoviruses That Harbors the ADP Protein and the Sodium Iodide Symporter Protein (NIS) under the Control of the Major Late Adenovirus Promoter

Functional organization of the major late transcriptional unit of canine adenovirus type 2

Vectors derived from canine adenovirus type 2 (CAV-2) are attractive candidates for gene therapy and live recombinant vaccines. CAV-2 vectors described thus far have been generated by modifying the virus genome, most notably early regions 1 and 3 or the fiber gene. Modification of these genes was underpinned by previous descriptions of their mRNA and protein-coding sequences. Similarly, the construction of new CAV-2 vectors bearing changes in other genomic regions, in particular many of those expressed late in the viral cycle, will require prior characterization of the corresponding transcriptional units. In this study, we provide a detailed description of the late transcriptional organization of the CAV-2 genome. We examined the major late transcription unit (MLTU) and determined its six families of mRNAs controlled by the putative major late promoter (MLP). All mRNAs expressed from the MLTU had a common non-coding tripartite leader (224 nt) at their 5' end. In transient transfection assays, the predicted MLP sequence was able to direct luciferase gene expression and the TPL sequence yielded a higher amount of transgene product. Identification of viral transcriptional products following in vitro infection confirmed most of the predicted protein-coding regions that were deduced from computer analysis of the CAV-2 genome. These findings contribute to a better understanding of gene expression in CAV-2 and lay the foundation required for genetic modifications aimed at vector optimization.

Central forkhead domain of human TFIIEβ plays a primary role in binding double-stranded DNA at transcription initiation

The human general transcription factor, TFIIE, consists of two subunits, alpha and beta. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIEbeta. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIEbeta binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function.

Histone N-terminal Tails Interfere with Nucleosome Traversal by RNA Polymerase II

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97 degrees bend in DNA AdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP-DNA AdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH 2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBP wt-DNA TATA function and structure depend both on the TATA box sequence and on the TBP species.

Syncytia formation affects the yield and cytotoxicity of an adenovirus expressing a fusogenic glycoprotein at a late stage of replication

Fusogenic membrane glycoproteins (FMGs) may enhance the cytotoxicity of conditionally replicative adenoviruses. However, expression at early stages of infection impairs virus replication. We have inserted the hyperfusogenic form of the gibbon ape leukemia virus (GALV) envelope glycoprotein as a new splice unit of the major late promoter (MLP) to generate a replication-competent adenovirus expressing this protein. At high multiplicity of infection (MOI), this virus replicated efficiently forming clumps of fused cells and showing a faster release. In contrast, at low MOI, infected cells formed syncytia where only one nucleus contained virus DNA, decreasing total virus production but increasing cytotoxicity.

G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

The transcription-repression domain of the adenovirus E1A oncoprotein targets p300 at the promoter

Extensive mutational/functional analysis of the transcription-repression domain encoded in the N-terminal 80 amino acids of the adenovirus E1A 243R oncoprotein suggests a model for the molecular mechanism of E1A repression: E1A accesses transcriptional co-activators such as p300 on specific promoters and then interacts with TBP to disrupt the TBP-TATA complex. In support of this model, as reported here, a basal core promoter activated by tethering p300 is repressible by E1A at the promoter level as shown by chromatin immunoprecipitation (ChIP) analysis. Sequestration of p300 by E1A does not play a significant role, as indicated by dose-response measurements. Furthermore, when the core promoter is transcriptionally activated by tethering activation domains of several transcription factors that can recruit p300 (p65, MyoD, cMyb and TFE3), transcription is repressible by E1A. However, when the core promoter is activated by factors not known to recruit p300 (USF1 and USF2), transcription is resistant to E1A repression. Finally, tethering p300 to the non-repressible adenovirus major late promoter (MLP) renders it repressible by E1A. ChIP analysis shows that E1A occupies the repressed MLP. These findings provide support for the hypothesis that p300 can serve as a scaffold for the E1A repression domain to access specific cellular gene promoters involved in growth regulation.

Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression

The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

GCV modulates the antitumoural efficacy of a replicative adenovirus expressing the TAT8-TK as a late gene in a pancreatic tumour model

Replication-competent adenoviruses carrying the herpes simplex thymidine kinase (TK) gene have shown contradictory evidence with regard to their antitumoural efficacy in combination with ganciclovir (GCV) treatment. We generated a replication-competent adenovirus carrying Tat8-TK, a modified form of the TK gene, under the control of the adenoviral major late promoter (AdRGDTat8-TK-L). Pancreatic cancer cell lines with different sensitivity to the TK/GCV system were infected with AdRGDTat8-TK-L, both in the presence and absence of GCV, and tested for treatment efficacy. We observed that, although the presence of GCV reduced viral replication in all infected cell lines, in three out of four GCV significantly enhanced the efficacy of the virotherapy. Interestingly, the cytotoxicity of the AdRGD-Tat8-TK-L/GCV was found more potent than that of a first generation AdTK/GCV system. In tumour xenografts from BxPC-3 and NP-18 pancreatic cells, both AdRGDTat8-TK-L and AdRGDTat8-TK-L/GCV treatment showed antitumoural activity. In BxPC-3 tumours scheduling of virus and prodrug was a key factor to determine the outcome of the therapy. Importantly, the addition of GCV enhanced the antitumoural effect of AdRGDTat8-TK-L only when applied in two rounds of virus+GCV. Interestingly, in spite of interfering with viral replication in vitro, GCV treatment of NP-18 tumours did not compromise the antitumoural efficacy of the AdRGDTat8-TK-L adenovirus. Thus, our results show that the combination therapy of a replicative adenovirus and the Tat8-TK/GCV suicide system can prove beneficial, when the appropriate regimen of virus and GCV is applied.

A Sequence Motif in the Simian Virus 40 (SV40) Early Core Promoter Affects Alternative Splicing of Transcribed mRNA

To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.

Epigenetic Status of an Adenovirus Type 12 Transgenome upon Long-Term Cultivation in Hamster Cells

The epigenetic status of integrated adenovirus type 12 (Ad12) DNA in hamster cells cultivated for about 4 decades has been investigated. Cell line TR12, a fibroblastic revertant of the Ad12-transformed epitheloid hamster cell line T637 with 15 copies of integrated Ad12 DNA, carries one Ad12 DNA copy plus a 3.9-kbp fragment from a second copy. The cellular insertion site for the Ad12 integrate, identical in both cell lines, is a >5.2-kbp inverted DNA repeat. The Ad12 transgenome is packaged around nucleosomes. The cellular junction is more sensitive to micrococcal nuclease at Ad12-occupied sites than at unoccupied sites. Bisulfite sequencing reveals complete de novo methylation in most of the 1,634 CpGs of the integrated viral DNA, except for its termini. Isolated unmethylated CpGs extend over the entire Ad12 integrate. The fully methylated transgenome segments are characterized by promoter silencing and histone H3 and H4 hypoacetylation. Nevertheless, there is minimal transcriptional activity of the late viral genes controlled by the fully methylated major late promoter of Ad12 DNA.